receptor signal transduction protocols

1 Radioligand-Binding Methods for Membrane Preparations and Intact Cells Mary Keen Introduction Radiollgand binding is a straightforward technique that measures the bindmg of a labeled agonist or antagonist to its receptor It is applicable to a variety of receptor preparations, ranging from purified receptors to tissue slices, or even whole animals However, membranes or broken-cell preparations are undoubtedly the most widely used Radioligand binding allows the affinity of drugs for their receptors to be determined very readily and It also allows the number of receptors in a tissue or cell to be quantified-something that was impossible before the mtroductlon of binding techniques Furthermore, the technique can be adapted to study the association and dissociation kinetics of llgand binding (1,2), as well as complex allosteric mteractlons between ligands (2) or between receptors and effector molecules, such as guanine nucleotidebinding proteins (G proteins) (2,2) Despite the ease with which radloligand binding can provide information regarding a wide range of receptors, it does have its limitations There is an absolute requirement for a high-affinity radioligand, selective for the receptor of Interest Even if such a radioligand exists, it may still be impossible to detect receptors in a particular tissue if receptor abundance is low relative to the nonspecific binding of the radioligand As with any technique, radioligand-binding data can be beset with artifacts if experiments are not designed carefully Most importantly, it must always be remembered that radioligand-binding experiments identify ligand-binding sites, which may or may not represent bonafide receptors These points are discussed m more detail m refs 1-3 This chapter provides detailed instructions on how to obtain and analyze equilibrium-binding data for the IP prostanoid (prostacyclin) receptor in human From Methods m Molecular Bralogy, vol 83 Receptor S/gnal Transducbon Edlted by R A J Chalks Humana Press Inc Totowa, NJ Protocols Keen platelets and the neuroblastoma x glioma cell line, NGlO%15, using the labeled agonist, [3H]-iloprost, as a radioligand This system illustrates many of the more common problems inherent in the design and analysis of radioligand binding experiments, because [3H]-iloprost is a rather difficult ligand Materials Platelets: Bags of frozen time-expired human platelets may be obtained from the Blood Transfusion Service (London, UK) and stored at -70°C until required NG108-15 cells: Confluent NG108-15 cells (see Note 1) are harvested by agitation in phosphate buffered saline and the cells pelleted by a low-speed spin (200g for mm) The pelleted cells may then be stored at -70°C until required Lysis buffer: mM Tris-HCI, pH 7.4 Wash buffer: 50 mMTris-HCl, 0.25 mM EDTA, pH 7.4 Assay buffer: 50 mA4Tris-HCI, mMMgCl,, pH 7.4 Note: Items 3-5 may all be prepared in liter quantrties and stored at 4°C until required [3H]-iloprost may be obtained from Amersham International, Amersham, UK The stock should be stored at -20°C and diluted in assay buffer on the day of the experiment Iloprost: Unlabeled iloprost is supplied with [3H]-iloprost from Amersham International or Schermg AG, Berlin, Germany The stock concentrations supplied may be stored at 4OC, being diluted m assay buffer on the day of the experiment Cell harvester and filters: A 24-place Brandel cell harvester and GF/B filters were used in the experiments presented here Both cell harvester and filters may be obtained through Semat Technical, St Albans, UK 5’-Guanylylimidodiphosphate (GppNHp [Sigma, Poole, UK]): 2.5 mMGppNHp (to give a final concentration of 100 pM) may be made up in I-mL quantities in assay buffer on the day of the experiment and stored on ice Any excess may be frozen at -2O’C and used in a subsequent assay 10 Curve-fitting programs: A wide range of suitable programs are available, but a detailed comparison is beyond the scope of this chapter However, it is useful to choose a program that allows parameters to be constrained to particular values, user-defined equations to be entered, and publication-quality output to be produced, The analysis and figures reproduced here were obtained using FigP (Biosofi, Cambridge, UK) Methods 3.1 Preparation of Platelet Membranes Thaw the bag of frozen platelets in cold water Centrifuge at 80,OOOg for 35 mm at 4°C Discard the superuatant Resuspendthe pellet in roughly 10 mL/U platelets of ice-cold mA4Tris-HCl, pH 7.4 Lyse any intact platelets by either stirring the suspensionon ice for 30 or freezing the suspension overnight and then thawing Use the most convenient method Radioligand-Binding Methods 3 Centrifuge the lysed platelets at 80,OOOg for 30 at 4’C Discard the supematam and resuspend the pellet in a convenient volume of ice-cold wash buffer Repeat step once Centrifuge at 80,OOOg for 30 at 4°C Resuspend the pellet in wash buffer to a concentration of about 20 mg protein/mL.‘As a rough guide, mL of pelleted membranes is equivalent to about 100 mg protein Freeze the final suspension in 1-mL aliquots at -70°C until required On the day of the binding assay, thaw the frozen membranes and dilute 1: 10 with assay buffer Do this a few minutes before startmg the binding assay, so that the membrane suspension just has time to come up to room temperature before pipeting (see Note 2) 3.2 Preparation of NG108-15 Cell Homogenates On the day of the binding assay, thaw the pelleted NGl08- 15 cells and resuspend in assay buffer, using 20 strokes of a tight-fitting glass Dounce homogenizer (Jencons, Leighton Buzzard, UK) or something similar The pellet from an 80-cm2 cell culture flask homogenized in about mL assay buffer provides sufficient homogenate for a single binding curve in thrs system Prepare the homogenate a few minutes before starting the bmding assay, so that the suspension has Just enough time to come up to room temperature before pipetmg (see Note 2) NG108-15 cells may also be prepared as washed homogenates (see Note 3) or whole cells (see Note 4) 3.3 Direct (Saturation) Binding This type of assay measures the equilibrium binding of a range of concentrations of the radioligand As no radioligand binds exclustvely to its receptor, it is also necessary to determine the nonspecific binding of each concentration of radioligand by suppressing its binding to the receptor by the inclusion of a saturating concentration of an unlabeled ligand (see Note 5) Specific binding can then be calculated as the difference between total and nonspecific binding (see Section 3.5.) The design of the binding experiment depends largely on the method to be used in separatingbound and free ligand (see Note 6) In the experimentspresented here, a Brandel cell harvester was used, which filters 24 samples simultaneously Therefore, each binding curve was designed to use 24 samples; concentrations of [3H]-iloprost (0.3-100 nM; see Notes and 8), each prepared in the absence and presence of 10 pM unlabeled iloprost, to define nonspecific binding (see Note 5), with eachdetermination being performed in duplicate Prepare appropriate dilutions of all ligands, and so on, in assay buffer (see Note 9) Pipet the assay components into polypropylene assay tubes (see Note 10) as follows: 30 pL assay buffer, 10 pL [3H]-iloprost, 10 pL assay buffer or iloprost, and 200 & membranes Adding the components in this order minimizes the risk of cross-contamination(seeNote 11) Keen 4 Following the addmon of the membranes, mix each sample well, and leave at room temperature for sufficient time for equilibrium to be achieved, m this case, 30 (see Note 12) Reserve leftover membranes for protein determination, and dilutions of radioligand for countmg (see step 8, below) If using an unfamiliar radiohgand, it is advisable to perform filter blanks, m which the total and nonspecific bmdmg of the radiollgand is determined m the absence of any membranes (see Note 13) At the end of the incubatton period, separate bound llgand from free ligand In this case, filter the samples onto GF/B glass fiber filters using a Brandel cell harvester and rapidly (see Note 14) rinse the filters with x 3.5 mL cold buffer (see Note 15) Place the filters m 5-mL scmtillatton vials (see Note 16) and add mL emulsifying scmtrilant Leave the vials overnight to allow the radiohgand to be extracted from the filters into the scintillant (see Note 17) Prepare radioligand standards Pipet 10 pL of each radroligand dilution mto 5-mL scmtillatron vials and add mL scmttllant These provide an accurate estimate of the actual amount of radtoligand added to each sample Count the samples and standards m a scmtillation counter 10 This basic method can be easily modified to examine the effects of modulators on radiohgand bmding Thus, to examme the effects of 100 p&Y GppNHp on [3H]-tloprost binding, as here, mitially add only 20 pL assay buffer to each tube and then add 10 p.L GppNHp The rest of the assay components are then added as described in step 3, above 3.4 Competition (Displacement) Binding In a competttion-bindmg assay,the bmding of an unlabeled ligand is measured by its ability to displace the specific binding of a low, fixed concentration of radioligand This is an extremely useful and versatile techmque, allowing the properties of a wide range of ligands to be investigated rapidly, even those with rather low affinities for the receptor; it 1swidely used as a primary screening technique in drug-discovery programs Furthermore, as the selectivity of the technique is determined by the selecttvtty of the radiohgand, it allows the study of the binding of nonselective hgands to one particular receptor of interest The design of the experiment again depends on the chotce of separation method In the case of a 24-place cell harvester, as used here, it is convenient to set up each binding curve as follows: nM [3H]-iloprost alone, to determine total binding (see Note 18); nA4 [3H]-iloprost plus 10 concentrations of unlabeled ligand (see Note 19), m the case of the experiments presented here, 0.1-3 pA4 iloprost; and4 nA4[3H]-iloprost plus 10pMunlabeled iloprost to define nonspecific bindmg (each determmation being performed in duplicate) Dilute the ligands and perform the assay exactly as descrtbed for dtrect bmdmg (see Section 3.) Remember to count 10 pL of the radioligand dilution as a concentration standard and retain excess membranes for protein determmation Radioligand-Binding Methods 3.5 Data Analysis A potential danger of radioligand binding is that the very simplicity of the technique may obscure the need for careful experimental design and thoughtful analysis of the data It IS easy to obtain reproducible data, feed it into a computer, and get values out It is much harder to be sure that those values really mean what you hope they mean As with all data analyses, the principle of rubbish in, rubbish out applies; if the data are highly scattered or riddled with artifacts, any estimates of receptor number or affimty generated by even the most sophisttcated of curve-fittmg techniques will be flawed A detailed consideration of the avoidance of potential artifacts is beyond the scope of this chapter, but is considered in some detail elsewhere (1,3) However, even if the data that go into the analysis are perfectly good, it is still possible to get rubbish out The hazards of using linearizations of bmdmg data, such as the Scatchard plot, are widely recognized (4,5), and these techniques should be avoided However, the use of nonlinear, curve-fitting techniques also has problems; as the number of variable parameters in the various models of binding increases, the number of combinations of these values that will fit the data more or less equally well also increases greatly There is no guarantee that the purely mathematical, nonlinear regression analysis will automatically converge on the biologically correct combination It is thus helpful to consider this sort of analysis as testing a hypothesis (se> Note 20) rather than automatically yielding precise and meaningful values For effective curve fitting, it is necessary to start with simple models, only increasing their complexity if the data demand; keep the underlying assumptions of the various models m mind, checking that the analysis gives sensible results; and check the results of the analysis for consistency with results from other experiments In order to illustrate these principles, the following sections consider in some detail the processes involved m analyzing some real data-the binding of [3H]-iloprost to platelets and NGlO8-15 cells In the case of [3H]-iloprost, analysis of competition-binding curves is somewhat more straightforward than direct binding, so these will be dealt with first 3.5.1 Analysis of Competition-Binding Curves I Calculation of radioligand concentration The precise amount of tracer radioligand included in eachsampleis determinedfrom the lo-& standard, follows: as radiohgand (pmol/sample)= (standarddpm)/(specific activity x 2220) where the specific activity of the radioligand is expressedasCMnmol and 2220 is a conversion factor This amountcan beconverted to a concentrationby taking the samplevolume into account.In this case,10 pL radioligand was included in Keen F 100 - F ma IE" a e 50 - s P P c c z ooI I I 1 o-l0 1o-g IO8 lo-' [iloprost] I I IO9 1o-6 (M) Fig K The inhibition of the specific binding of 7.3 nA4 [3H]-iloprost by unlabeled rloprost m the absence (A) and presence (8) of 100 pi14 GppNHp in human platelet membranes The solid line represents the best fit of a single-site model of binding to the data obtained in the absence of GppNHp; the dotted line represents the best fit of the same model to the data obtained in the presence of GppNHp (see Section 3.5.1 for details) a final assay volume of 250 & Thus the radioligand concentration in nM(=pmol/mL) is given by: [radioligand] (nM) = (standard dpm x 4)/(specific activity x 2220) Calculation of % inhibition of specific binding The data presented here (Figs and 2) are expressed as % inhibition of specific binding of [3H]-iloprost, which IS calculated as follows: % inhibition = 100 x { I- [(sample dpm - nsb dpm)/(total dpm - nsb dpm)]) where sample dpm is the mean dpm in each sample containing a particular concentration of the unlabeled lrgand, total dpm is the mean dpm in the samples containing [3H]-rloprost alone, and nsb dpm is the mean dpm in the samples containing 10 p.M unlabeled rloprost to define nonspecific binding This transformation of the data IS useful in that it incorporates total and non- Radioligand-Binding Methods specific binding into the inhibition curve so that low concentrations of unlabeled ligand should inhibit 0% specific binding and high concentrations inhibit 100% However, the transformation is by no means essential and may incorporate errors, if estimates of total and nonspecific binding are not accurate (see Note 1) The effect of GppNHp on iloprost binding to platelet membranes a The inhibition of the specific bindmg of 7.3 nA4 [3H]-iloprost by unlabeled iloprost in the absence and presence of 100 pM GppNHp is shown m Fig The first step in the analysis of any binding data should be to determine the best fit of the simplest model of binding to the data: the simple Langmuir isotherm, or single-site model This model assumes binding to a single population of noninteracting sites, so that: WI = &a, P-W + PI) where [LR] is the concentration of ligand-receptor complexes (equivalent to % inhibition in this case), B,,, is the maximal binding capacity or total number of receptor sites (which should be 100% inhibition in the case of competition binding data), and K is an estimate of ligand-binding affinity (see part c, below) Curve fitting is performed by computer assisted, nonlinear regression analysis A wide range of suitable programs are available (see Section 2.) Fitting the single-site model to the binding curves in Fig yielded the following estimates for K and B,,,: K B max no GppNHp 2.00 x 10-8M 99.7% + GPPNHP 2.11 x l&s44 100.8% b Inspection of the predicted curves plotted in Fig shows that the curves tit the data rather well Furthermore, the estimates of K and B,,, seem sensible; in each case B,,, is close to 100%, as expected, and the estimates of K obtained m the absence and presence of GppNHp are very similar, in accord with the observation that GppNHp appears to have very little effect on iloprost binding under these circumstances (see Fig 1) Thus the fit of the single-site binding model to the data seems satisfactory, and no further curve fitting IS required (or justified) in this case c Correction for the presence of [3H]-iloprost Competition-binding curves, such as those shown in Fig 1, are necessarily obtained in the presence of a low, fixed concentration of radiohgand The presence of this radioligand affects the position of the binding curve for the unlabeled ligand; it is shifted to the right of its true position by a factor determined by the radiohgand aftinity and concentration Thus, it is necessary to correct estimates of affinity obtained from competition studies to take this rightward shift into account, using the Cheng-Prusoff equation (6): true Kd = K/{ + ([L*]/K*)} IA 100 50 00 I 1O“O I I 1o-g IO9 [iloprost] I IO.' I I IO" 1o-5 (M) IB A I I I 1O"O lo-g 1o-6 lo-' [iloprost] (M) I I I 1O-e 1O'5 Fig The inhibition of the specific binding of 2.4 nM [3H]-iloprost by unlabeled iloprost in the absence (A) and presence (V) of 100 @4 GppNHp in NGl08-15 cell homogenates The lines represent the best fit of various models to the data (A) The dotted lines represent the best fit of a single-site model of binding; the solid lines represent the best fit of the same model, in which B,,, is constrained to 100% (B) The solid lines represent the IC g - IOO- E z % 50 - s tl P $ O0 I lo-I0 I losg I IO-* [iloprost] 1o-7 I I 1o-5 lo-s (M) ID ;z 100 - P I * I lo-lo I 1o-g A I I I I IO4 1o-7 lo+ 1o-5 [iloprost] (M) best fit of the Hill equation to the same data (C) The solid lines represent the best fit of a two-site model of binding (D) The solid lines represent the best fit of a single-site model of binding to the data obtained in the presence of GppNHp and the best fit of a two-site model of binding to the data obtained in the absence of GppNHp, in which K2 has been constrained to the value of K estimated in the presence of GppNHp (see Section 3.5.1 for details) 10 Keen where true Kd IS the corrected dissociation constant for the unlabeled ligand, [L*] is the radioligand concentration (determined from the radioligand standard; see step 1, above), andK* is the dissociation constant for the radioligand, determined from a direct-binding assay (see Section 3.5 ) In the case of a self-competition assay, as here, where the same ltgand is used as both radioligand and competing ligand, the Cheng-Prusoff equation simplifies to: true Kd = K - [L*] with the assumption that the affinities of the labeled and unlabeled form of the ligand are the same Thus, for the iloprost binding to platelet membranes Kd no GppNHp 1.27 x 10-8M + G~PNHP 1.34 x 10-Q! The effect of GppNHp on iloprost binding to NG108- 15 cell homogenates: a The inhibition of the specific binding of 2.4 nM[3H]-iloprost by unlabeled iloprost in the absence and presence of 100 @fGppNHp is shown in Fig 2A Again, the first step in the analysis of these data should be to determine the best fit of a smgle-site model to the data This yielded the followmg estimates for K and B,,,, no GppNHp K Bmax 6.06 x 10-gM 96.2% + GPPNHP 199x10-8M 95.2% b While the predicted curves fit the data points reasonably well, and the estimated K values reflect the fact that the curve obtained m the presence of GppNHp lies to the right of the curve obtained in its absence (see Fig 2A, dotted lines), both estimates of B,,, are less than the expected value of 100% Thus, the sensible next step m the analysis is to see if these data are consistent with a B,,, value of lOO%, by fixing B,, to this value: K Bmax no GppNHp 6.90 x 10-9A4 100% (fixed) + GPPNHP 2.31 x lVsh4 100% (fixed) The predicted curves given by this procedure are shown in Fig 2A (solid lines) The curves still fit the data well and are thus likely to represent a better estimate of K than those obtained when B,,.,,,is allowed to converge on a value of less than 100% (see Note 22) c The close agreement between the data and the single-site binding curves may suggest that any further analysis of this data using more complex models is unJustitied However, the rightward shift in the binding curve obtained in the presence of GppNHp is characteristic of G protein-coupled receptors It seems to reflect the ability of the guanme nucleottde to disrupt receptor-G protein complexes (RG), which have high affinity for agonists, converting them to uncoupled receptors (R), which have low agomst affinity If the results of the lredale et al 258 and then transferred to a solid filter The filter is then hybridized with the radiolabeled probe and the specific RNA band is visualized by autoradiography There are some advantages of the Northern blot technique over the RNase protection assay First, Northern blot is a technically more simple protocol Second, Northern blot analysis allows you to visualize the full-length mRNA species, to confirm that the probe is hybridizing with the specific mRNA of interest Even if the majority of the RNA studies will be conducted by RNase protection, it is important to confirm the specificity of the riboprobe by Northem blot analysis Third, both random prime-labeled DNA probes and riboprobes can be used for Northern blot analysis 3.1.1 RNA Isolation For preparation of RNA from tissue: The tissue should be removed quickly from the animal and frozen immediately on dry ice (see Note 1) Polytron frozen tissue in mL of GIT in a 50-mL tube (see Note 2), and centrifuge at 500g to reduce foam Alternatively, incubate tubes on ice until foam dissipates For preparation of RNA from cultured cells: The media is removed and mL of GIT is added to the culture dish containing -1 x 1O6cells Carefully transfer GIT/RNA solution to ultracentrimge tubes containing mL cesium chloride Add GIT until tubes are filled to approx cm from the tube rim Place tubes in ultracentrifuge buckets and balance all tubes to the same weight with GIT Centrifuge samples at 150,OOOg at 22°C for h Aspirate or decant supernatant, and wipe remaining liquid from walls of tubes using Kimwipes (see Note 3) Thoroughly resuspend the RNA pellet in 360 pL DEPC-treated water by repeat pipeting (see Note 4) Transfer RNA to a microcentrifuge tube Add 40 pL of 3M sodium acetate (RNase-free) to each sample and mix 10 Add 2.5 vol(lOO0 pL) ice-cold 100% ethanol to samples, vortex, and either place on dry ice for 30-45 or precipitate overnight at -2OOC 11 Spin for 15-30 at 17,000g at 4°C 12 Remove supernatant and resuspend pellet in 70% ethanol 13 Spin RNA for at 17,000g at 4’C 14 Remove supernatant, air dry RNA, and resuspend in DEPC-treated water 15 Quantitate RNA at OD,,, (OD,,, l= 40 ng RNA/l pL) (see Notes and 6) 16 Store RNA at -80°C 3.1.2 Riboprobe Template Preparation In an autoclaved microcentrifuge restriction enzyme buffer, 40-60 water to a final volume of 100 pL Incubate in water bath for 3-5 h tion enzyme tube, mix 10 pg of plasmid DNA, 10 pL of U of restriction enzyme, and DEPC-treated (see Notes and 8) at temperature recommended for the restric- Regulation of Receptor Expression 259 Add pL 10% SDS and p.L mg/mL Proteinase K and incubate for 45 at 37°C Add 140 pL DEPC-treated water and extract twice with 250 pL phenol/chloroform/ isoamylalcohol Precipitate DNA: To the aqueous phase, add 25 cls, 3M sodium acetate and 700 pL cold 100% ethanol Mix thoroughly by inversion and precipitate on dry ice for 20 Centrifuge for 20 at 17,OOOg at 4°C Wash pellet in cold 70% ethanol and centrifuge for at 17,000g at 4OC Remove ethanol and lyophilize pellet Resuspend pellet in 40 pL DEPC-treated water (approximate DNA concentration is now 250 ng/&) 10 Run pL DNA on 1% agarose gel to verify completeness of digestion and approximate percent recovery of DNA 11 Store DNA template at -20°C 3.7.3 Riboprobe Transcription Warm to room temperature: 5X transcription buffer, 0.75M dithlothreitol, premixed nucleotides, DNA template, and [c+~~P]-NTP (see Notes 9-l 1) In an autoclaved mlcrocentrifuge tube, add pL transcription buffer, pL dithiothreitol, pL premixed nucleotides, p.L RNase block, pg DNA template, pL [cz-~~P]-NTP, pL undiluted polymerase (see Note 12), and enough DEPC-treated water for a total volume of 25 pL Make sure to add the polymerase last Pulse spin the sample in a microcentrifuge to mix and remove bubbles, and then incubate for h at 37°C Destroy the DNA template by adding 10 U of DNase and incubating for 15 at 37°C (see Note 13) Inactivate the enzymes by adding 1.3 @-.0.5M DEPC-EDTA Add DEPC-treated water to a final volume of 70 $ and column puni@ on an STEequilibrated NUCTRAP push column, according to manufacturer’s instructions Count the eluate to determine counts per minute Store probe at -2O’C and use within d 3.1.4 Random Primed Labeling of DNA DNA fragment to be random primed labeled must be gel purified There are numerous methods and kits to accomplish this, which will not be discussed here (see Notes 15-l 7) The Compass DNA purification kit (American Bioanalytical) is highly recommended The DNA (50-100 ng) dissolved in distilled water must be denatured at 100°C for 5-10 min, followed by quick-cooling on ice Reaction mix is added to the tube to final concentrations of 0.2MHEPES, 50 mM Tris-HCl, @4 MgC&,lO mM BME, 0.4 mg/mL BSA, 5.4 ODzao U/mL oligodeoxyribonucleotide hexamer primers, pH 6.8 (Gibco reaction mixture) lredale et al 260 The cold nucleotides (except for dNTP chosen for radioactive labeling) are added to a final concentration of 20 fl each Bring reaction mix up to a volume of 44 pL with distilled water Add pL of the [a-32P]-dNTP and mix Add Klenow fragment (1 pL or U) to the tube Incubate reaction for h at room temperature Stop reaction by heating to 65°C for 10 or by addition of NEDTA 10 Purify the probe on a Nick column according to the manufacturer’s protocol Il Count an aliquot of the probe to determine the amount of incorporated radloactivity I RNA Formaldehyde Gel Preparation of gel (see Notes 18-20): MIcrowave at medium setting 0.9-1.2% agarose in 1X MOPS until the agarose is in solution When the flask is cool enough to grasp, add pL of ethidium bromide, a final concentration of 2% formaldehyde (6.2 mL of a 37% formaldehyde solution), and double-distilled autoclaved water to a total volume of 115 mL In a fume hood, pour mixture into casting tray with combs and allow to solidify (>45 mm) Thaw RNA on ice, aliquot into RNase-free tubes, and lyophilize Resuspend RNA pellets in 10-15 pL of sample buffer, heat to 99°C for mm, flick samples, and heat for another minute Pulse spin tubes in microfuge Place gel in electrophoresis tank filled with 1X MOPS, and load the RNA samples mto the gel wells and sample buffer into the unused wells (see Note 1) Run the gel at 60-80 V for 4-5 h, or until the bromophenol dye is 3/4 through the gel (see Note 22) Place the gel onto plastic wrap (see Note 23) and photograph on UV transllluminator to visualize the ribosomal RNA (28S, 18S, which correspond to approx and kb, respectively) This will also demonstrate the quality of the RNA (i.e., if degradation has occurred) Wash gel twice for 15 in 10X SSC, while gently shaking to remove the formaldehyde 3.1.6 Capillary Transfer of RNA to Solid Support Cut mtrocellulose and Whatman 3MM filters the same size as the gel Also, cut a wick the same width as the gel, but approx 30 cm long Set up gel transfer apparatus as follows (see Notes 24 and 25): Fill reservoir with 10X SSC; place wick on a platform suspended above reservoir and submerge both ends of wtck in reservoir; place gel on the wick with the open side of the wells facing down; prewet mtrocellulose in 2X SSC and place on top of the gel; prewet Whatman filter papers in 10X SSC and place on top of the mtrocellulose; add 16-20 absorbent filters, 11 x 14 cm (available from Gibco), or a stack of paper towels cut to the appropnate size; place glass/plastic plate on top of transfer apparatus; on top of plate place heavy objects, such as two 250~mL bottles, with approx 100 mL water in each Regulation of Receptor Expression 261 Transfer for approx 18 h Dismantle transfer apparatus and view nitrocellulose on transilummator With a pencil, mark the orientation of gel and location of ribosomal bands on the side of the nitrocellulose UV crosslink the nitrocellulose or bake it at 80°C for h Store blot m plastic wrap in the refrigerator or use immediately 3.1.7 Hybridization Immediately prior to hybridization, denature salmon sperm DNA for 10 at 100°C and quick-cool on ice Add DNA to the buffer at a final concentration of 100-200 pg/mL For hybridization in water bath: Place blot in a heat sealable pouch (16.5 x 20.3 cm) with 10-15 mL of hybridization buffer and denatured salmon sperm DNA Carefully seal and place in shaking preheated water bath 3, For hybridization in oven (see Notes 26 and 27): Place blot between two pieces of mesh and roll up from bottom to top Place mesh/blot in hybridization tube and add 15-20 mL of hybridization buffer Weigh and balance tubes and place in preheated rotating oven Prehybridization: l-2 h at 42’C (cDNA) or 65°C (riboprobe) Denaturation of probe: Heat riboprobes at 85°C for Heat cDNA probes at 95-100°C for Both should immediately be quick-cooled on ice before use Hybridization: Add probes to hybridization buffer with or without additional buffer (see Notes 28 and 29) Hybridization reaction should proceed overnight 3.1.8 Posthybridization Washes If washes are in hybridization oven, then pipet buffer out of the tube and dispose in liquid radioactive waste Add 2X SSC, 0.1% SDS heated to hybridization temperature (see Note 30) to the tube and place back in the rotating oven for 20 If hybridization was performed in a sealable pouch, carefully remove the blot from the pouch and place in a container for washing Discard remainder of the buffer in liquid radioactive waste Wash blot in several hundred milliliters of 2X SSC, 0.1% SDS at the hybridization temperature Additional washes (see Note 1): The remaining washes are in increasingly stringent conditions in shaking water baths Riboprobe hybridized blots are washed successively in 2X SSC, 0.1% SDS at 65”C, 0.5X SSC, 0.1% at 65’C, and 0.1X SSC, 0.1% SDS at 68°C Blots hybridized with cDNA probes are washed in 2X SSC, 0.1% SDS at 55’C, 0.5X SSC, 0.1% SDS 55°C and 0.1X SSC, 0.1% SDS 58°C Wash at each condition for 20-30 and check radioactivity remaining on blots after each wash, with a hand-held counter Cover blot in plastic wrap and expose to film Store blot in plastic wrap for hybridization with additional probes The method of quantitation depends on the equipment available in the laboratory Phosphoimagers work well for RPA and Northern blot quantitation and produce publishable quality images Exposure to film is also important after analysis lredale et al 262 to preserve a hard copy of the image Another possibility is to quantitate an autoradiogram using an image-analysis system and the National Institutes of Health image analysis program Normalize data with the internal standard 3.2 The RNase Protection Assay The RNase protection assayutilizes a radiolabeled rlboprobe that is complimentary to the RNA of interest Hybridization of the riboprobe with total RNA is conducted in solution, and the sample is subsequently treated with RNase to degrade unhybridized probe Only the specific RNA:RNA hybrids are resistant to RNase treatment, so that nearly all nonspecific background is removed The protected RNA hybrids are then visualized by autoradiography There are several advantages of the RNase protection assay Most importantly, it is more sensitive than the Northern blot and therefore allows for analysis of rare mRNA species The other major advantage 1sthat it 1svery specific and there is very low background because of the posthybridization RNase treatment However, the RNase protection assay 1smore technically difficult A very specific probe must be used, which means that in most cases the sequence of the riboprobe must be derived from the same species as that of the RNA to be analyzed If the sequence of the riboprobe is not identical, the mismatched regions of the probe will be susceptible to RNase digestion You should also verify that the sequence that you have chosen is specific for the receptor of interest For example, the transmembrane regions of many receptors within a family can be very similar The riboprobe should also be of an appropriate length (i.e., -lOO500 bp), as larger probes are susceptible to breakdown and will yield multiple bands on a gel 3.2.1 Probe Preparation Label and purify receptor-specific riboprobe according to instructions in Section 3.1.3 Use only riboprobes with a percent incorporation greater than 30% (see Notes 32 and 33) Dilute an aliquot of probe to x lo5 counts/min/& m DEPC-Hz0 and store at 4°C The remainder of the probe should be stored at -20°C 3.2.2 End-Labeling DNA Size Markers In a total volume of 20 $, mix a DNA size markers and & 1OX Klenow &zyme buffer (see Note 34) Heat DNA for at 8O’C Add 10 @i of appropriate [a-32P]-dNTP and U Klenow enzyme Incubate for 10 mm at room temperature Precipitate the labeled DNA by adding an equal volume of 4M ammonium acetate and to the new volume, 2.5 vol of Ice-cold 100% ethanol Place on dry Ice for 20 Regulation of Receptor Expression 10 263 Centrifuge at 17,000g for 20 mm at 4’C Rinse pellet in cold 70% ethanol and lyophilize Resuspend in 50 pL double-distilled deionized H,O Count & and dilute small aliquot to 1000 counts/min/pL Store diluted and undiluted aliquots at -20°C Markers are usable for several weeks 3.2.3 Sample Hybridization Lyophilize RNA samples in 1.5~mL autoclaved microfuge tubes and resuspend completely in 29 $ hybridization buffer Add pL of diluted probe to each sample and heat for 10 mm at 85°C Quickly transfer the samples to a 65°C water bath and hybridize 16-18 h 3.2.4 Denaturing Polyacrylamide Gel Preparation 1, For two 8% polyacrylamide mini-gels, add 1.2 g acrylamidelbu (19: 1) and 7.2 g urea (final concentration of 8M) to 15 mL 1X TBE m a 50-mL Falcon tube (see Notes 35 and 36) Shake the tube and heat at 65°C for at least 15 to dissolve solutes completely Cool the solution to approximately room temperature and filter through Whatman No filter paper into another 50-mL Falcon tube Add 90 pL 10% APS and pL TEMED to the tube and mix thoroughly without creating air bubbles Using a lo-mL syringe, pour the gel solution into each mini-gel casting apparatus Insert IO-well combs and allow gels to polymerize >2 h Just prior to use, run each apparatus under cold tap water to remove excess acrylamide from combs Place gels in buffer chamber and fill with 1X TBE accordmg to manufacturer’s instructions Within l-3 of sample loading, clear urea from each well using 1X TBE in a lo-mL syringe with a 20-gage needle 3.2.5 Sample Treatment To each hybridized sample, add 400 $ RNase digestion buffer with 25 pg/mL RNase A and 125 U/mL RNase Tl Mix thoroughly by inversion and incubate 45 at 37OC Add 20 @., 10% SDS and 50 pg Proteinase K and mix by inverting 5-7 times Incubate for 15 at 37OC Add 400 pL phenol/chloroform/isoamyl alcohol, vortex 10 s, and spin in microfuge for Transfer aqueous phase to new 1.5-mL tube and precipitate RNA as follows: add $ 10 mg/mL tRNA carrier and mL ice cold 100% ethanol and mix vigorously by inversion Place on dry ice for 20 Spin samples in microfuge for 20 at 17,000g at 4’C Wash pellets with 95% ethanol and spm m microfuge for at 4°C 264 /redate et al Remove ethanol and lyophilize samples Thoroughly resuspend pellets in 10 pL gel loading buffer 10 If radiolabeled DNA size markers are to be used, 1000 counts/mm of 32P-dNTP end-labeled DNA should be added to 10 $ loading buffer 11 Incubate all samples for at 95OC, pulse spin in microfnge, and quack-cool on ice 12 Load samples onto gels, and run at 200 V until xylene cyan01 dye front has run to the bottom of the gels (see Notes 37-40) 13 Fix gels in 15% methanol/5% acetic acid for 10 mm 14 Transfer gels onto Whatman MM Chromatography paper and cover with plastic wrap 15 Dry gels for 40 at 8O’C 16 Expose to X-ray film with an intenstfier screen overnight 3.3 Receptor Half-Life Studies The half-life of mRNA in cultured cells can be determined by measuring the decay of the mRNA in the presence of a DNA transcription inhibitor The mRNA can be measured by either Northern blot or RNase protection analysis Pretreat cells with agonist of interest for time period which shows maximum effect Add actinomycin D (2 mg/mL) to these cells as well as to control cells, harvest at 0.5, 1,2, 3, and h after addition of the transcription inhibitor (see Note 41) Perform RNase protection assays (see Sectton 3.2 ) on RNA isolated (see Section 2.2.1.) from these samples, as well as from control and agonist-treated cells not treated with actinomycm D Compare the rate of decay of the mRNA species of interest in control and agomst-treated cells (see Note 42) 3.4 N&ear Run-On The nuclear run-on assay provides a measure of the rate of gene transcription Cell nuclei are isolated from cultured cells andthen incubated with [cI-~~P]T.-JTP and unlabeled NTPs in order to label nascent RNA transcripts [a-32P]-labeled RNA IS then purified and specific RNA transcripts are detected by hybridization to cDNA that is immobilized on nylon or nitrocellulose membranes The level of hybridization to the cDNA IS a measure of the transcription rate This technique provides important information on the regulation of transcription rate, but it is technically very demanding (Don’t get discouraged if you don’t succeed the first time; it usually takes several attempts to obtain useful results.) 3.4.1 Preparation of CDNA and Gel Purification Isolate at least 500 ng of the plasmid the Qiagen Maxi Prep ktt containing the cDNA(s) of interest, using Regulation of Receptor Expression 265 Incubate plasmid overnight at 37°C with the appropriate restriction enzymes to excise the cDNA of interest from the vector Pour a 100~mL low-melt agarose gel containing 0.8, 1, or 1.5% agarose (dependent on the size of cDNA of interest), 0.01% (w/v) ethidium bromide in TAE Add DNA-loading buffer to the cut plasmid and load the low-melt agarose gel (see Note 43) Electrophorese the gel at approx 50-70 mV until there is complete separation of the cDNA of interest from the vector Locate the bands of interest with a UV transilluminator and use a sharp razor blade to cut them from the gel Trim away as much excess agarose as possible Place each slice mto a 1.5~mL Eppendorf tube and add vol of TE buffer, pH 8.0 Incubate at 70°C for to melt the agarose, and mix gently Quick-freeze the samples m dry ice for 30 10 Thaw the samples, tap gently, and centrifuge at 17,OOOg for 5-10 11 Remove the supernatant and ethanol precipitate the DNA by addition of l/l0 vol of 3M sodium acetate and vol of ethanol 12 Resuspend and combine the samples in -100 pL of TE buffer, pH 7.4 3.4.2 Preparation of CDNA Slot Blot Dilute the cDNA of interest to a final volume of 660 @, of TE buffer, pH 7.4 (for six samples), in a 1.5-n& Eppendorf tube Add 73.5 & of 1MNaOH Heat for 10 at 95°C and place on ice Add 7.4 pL of 6X SSPE and mix well Assemble a Bio-Rad Bio-Dot SF Microfiltration apparatus with six pieces of tilter paper (prewetted with 6X SSPE), with one piece of Biotrans nylon membrane on top (see Note 44) Attach the apparatus to a vacuum and apply -300 pL of 6X SSPE through each slot Apply 120 pL of cDNA to each slot (see Note 45) It is necessary to use at least pg of each cDNA Mark the locations of those slots containing DNA (see Note 46) Remove the nylon membrane and immediately UV crosslink 10 Air dry the blot overnight; the following morning bake for h at 80°C 11 Cut the membrane into strips, each containing the cDNA of interest and the control(s) cDNA (see Note 46) The blots are now ready for prehybridtzation 3.4.3 Prehybridization of the CD/VA Slot Blots Denature 1.2 mL of salmon sperm DNA (10 mg/mL) for 10 at 105’C Add mL of salmon sperm DNA to 49 mL of prehybridization solution and mix well Place each blot into a separate 50 mL screw-capped Falcon tube and add mL of prehybridization solution Prehybridize at 42°C for at least h in a rotating hybridization oven The blots are now ready for hybridization with the newly transcribed RNA from Section 3.4.5 266 tredale et al 3.4.4 Preparation of Nuclei for Nuclear Run-On It is better to prepare fresh nuclei on the day of the experiment than to rely on frozen samples Fresh nuclei can be generated as described by Klely et al (s) Wash the cell monolayers twice with ice-cold PBS Add mL of buffer I (see Note 47) to each loo-mm dish and keep on ice for 10 Add a further mL of buffer I containing 6il4 sucrose and 0.6% Triton X-100 and scrape the cells mto a chilled Dounce homogenizer (see Note 48) Homogenize by six strokes with a type A pestle and layer the homogenate over mL of 0.6M sucrose in buffer I in a 10-mL centrifuge tube (see Note 49) Centrifuge the tubes at 2000g for 10 at 4°C in a fixed-angle rotor Rapidly pour off the supematant by invertmg the tube, resuspend the pellet in 200 pL of glycerol buffer, and store on ice (see Note 50) Estimate the number of nuclei using a hemocytometer 3.4.5 Nuclear Run-On Transfer the nuclei (at least x 10’) to a 2-mL Eppendorf tube (see Note l), add 25 & (250 @i) of [cz-~~P]UTP followed by 200 & of RX mix, into a mL Eppendorf tube, and incubate for 30 in a 30°C shaking water bath Add 20 pL of DNase (RNase free; U/mL), followed by 4.6 pL of 100 mM CaCl, solution, and incubate for a further 10 mm at 30°C in the shaking water bath Add 37 & of PK mix and incubate for 10 at 37°C in the shaking water bath Shear the nuclei through a 26-gage 0.5 needle and split into Eppendorf tubes (2 mL) (see Note 52) To each tube add 450 cls, of GIT solution (with BME), 70 & of 2A4 sodium acetate, 800 pL of saturated phenol solution, and 155 & of chloroform/ isoamylalcohol(49: l), mix well, and incubate on ice for 15 Centrifuge for 15 at 17,OOOgat room temperature Caretilly transfer the aqueous layer to another 2-mL Eppendorftube and add & of tRNA (10 mg/mL), pL of glycogen (20 mg/mL), and an equal volume of propanol Mix well and centrifuge for 15 at 17,OOOgat room temperature, Remove the supematant, being careful not to disturb the pellet, and wash with 70% ethanol 10 Air dry or speed-vacuum dry the pellet, resuspend m 100 # of DEPC-treated water, and recombine the two pellets from each sample 11 Count pL in duplicate from each sample 12 Heat the samples to 95°C for and place on ice 13 Add an equal number of radioactive counts to each of the prehybrid blots from Section 3.4.3 (see Note 53) 14 Hybridize for 48 h at 42’C in a rotating hybridization oven 15 Wash the blots m 0.2% SSPE, 0.1% SDS for 10 at 42°C m a shaking water bath 16 Wash the blots m 0.1 % SSPE, 0.1 % SDS for up to 10 at 42’C in a shaking water bath (seeNote 54) Regulation of Receptor Expression 267 17 Dry the blots on filter paper at 37°C for approx 18 Cover the blots with Saran wrap and expose to film or a Phosphoimager 19 Quantitate using image analysis or phosphoimage analysis and normalize the signal from the cDNA of interest with the control cDNA Notes In order not to overload the GITKsCl density gradient, a maxlmum of g of brain tissue or l/2 g of peripheral tissue per ultracentrifuge tube should be used The tissue must be homogenized as quickly as possible to avoid degradation of RNA This can be achieved by freezing the tissue m small pieces (-50 mg), or by rapidly pulverizmg larger frozen tissue sections with a prechilled mortar and pestle Following CsCl centrifugation, the RNA pellet at the bottom of the tube will be both transparent and gelatinous The volume m which the final RNA pellet is resuspended depends on amount of RNA expected It is better to make concentrated samples that can be diluted following quantitation than to make samples that are too dilute to be useful These latter samples would have to be lyophilized or reprecipitated A large quantity of RNA should be diluted into several aliqouts to avoid freeze/ thawing unnecessarily To check the quality of the RNA, an OD 260,280 ratio of b1.7 is acceptable Values of ~1.7 may indicate protein contamination It is extremely important that the riboprobe template IS completely linearized prior to transcription Reagents used for template preparation must be RNase-free Contaminated or dirty template stocks are a common cause for low incorporation Make sure that the nucleotlde used in the riboprobe transcription reaction is a ribonucleotide and not a deoxyribonucleotide 10 CTP and UTP are recommended for radiolabeling riboprobes The choice of which one depends on the number of sites of incorporation for each nucleotide: the more sites, the higher the specific activity of the probe However, if the number of sites is too great, the radlolabeled RNA backbone may be unstable and probe degradation may occur 11 In some procedures requiring the use of a riboprobe, a full transcription reaction is not necessary In such cases, a half-reaction (i.e., half the normal volume of reagents in a total reaction volume of 12.5 pL) may be used The sample can then be DNase-treated and purified just as with a full reaction, but make sure the sample volume is 70 pL before loading it on the column 12 Some protocols warn against the use of even the slightest excess of polymerase in an RNA transcription reaction, because of the possibdity of nonspecific transcription initiation at other promoter sites in the plasmld However, this warning may be unfounded, since newer plasmids can contam multiple RNA polymerase promoters on the same strand of DNA without causing problems with transcription 13 The main source of background hybridization in techniques that use riboprobes, such as the RNase protection assay, may be the incomplete digestion of the DNA lredale et al 14 15 16 17 18 19 20 22 23 template following the transcription reaction Some of the newly synthesized probe may anneal to the complementary portion of the DNA template and, thus, render that region of the plasmid resistant to DNase treatment This DNA would then be column-purified with the riboprobe and, during a hybridization reaction, would compete with mRNA for the probe Resultant DNA-RNA hybrids would be resistant to RNase digestion and would increase the background of RPA samples on a polyacrylamide gel If such a scenario is suspected, then the following modification should be made to the riboprobe transcription procedure: Following the labeling reaction, the sample should be heated for 10 to 95’C for to denature DNA-riboprobe hybrids Following quick cooling on ice for 10 s, DNase could then be added in the usual manner Repeated freeze-thawing of reagents and synthesized riboprobes should be avoided, Aliquot materials in volumes that will not be freeze-thawed more than 3-5 times In the random prime labeling technique, it is very important to determine the specificity of the DNA fragment to be labeled The lack of probe specificity can cause unforeseen background hybridization problems A search through GenBank would be useful Another cause of high background hybridization when random prime probes are used is the presence of vector sequences in the fragment of interest A DNA fragment with vector sequence at its ends should be digested at internal restriction sites to remove this unwanted DNA The DNA fragment of interest must then be gel purified, making sure that the cleaved ends not comigrate with it The purification of multiple bands may result in hybridization to inappropriate target mRNAs Low incorporation of radioactivity is generally caused by the quality of the punfied DNA Although the purified DNA may appear clean on an agarose gel, impurities may be inhibiting the Klenow fragment If this happens, a quick clean up of the DNA may solve the problem Spin columns or phenol-chloroform extraction and ethanol precipitation are recommended Wash flasks, gel casters, combs, and gel apparatus m 6% H,Oz for 20 min, followed by a quick rinse in DEPC-treated water to ensure that they are RNase free prior to use If feasible, a gel apparatus and tank should be dedicated solely for RNA gels RNA sample amounts: The amount of RNA necessary for message detection is dependent on the relative abundance of the target mRNA Rare messages may require the isolation of mRNA in order to detect a signal This is often the case with G protein-coupled receptors Hence, RPAs are commonly used, instead of Northern blot analysis, because of then enhanced sensitivity The outermost lanes on either side of the gel should not be used if at all possible RNA in these lanes tend not to transfer as well Running the gel too fast may heat the agarose and distort the lanes of migrating RNA Prior to removing the gel from the tank, it is advisable to cut off a comer of the gel for orientation purposes Regulation of Receptor Expression 269 24 It 1s important to remove all air bubbles between layers of materials being added to the capillary transfer apparatus Air bubbles will prevent even transfer of RNA to nitrocellulose A disposable IO-mL serological pipet works well to roll out the bubbles 25 When prewetting mtrocellulose in 2X SSC, start with one comer and progress slowly until the entire piece is wet This will prevent trapping of air in the membrane 26 A hybridization oven is preferable to heat sealable bags for their ease m working with radIoactivity There is less likely to be problems with leaking buffers and contamination of water baths or equipment 27 If using hybridization tubes, be sure that the blots/mesh are not unraveling Check this during the prehybridizatlon step 28 The amount of labeled probe added to the hybridization buffer depends on the relative abundance of the target RNA A good startmg point IS x lo6 counts/min/mL buffer for abundant clones and 2-3 x IO6 countslminlml for rare messages 29 Internal standards: Quantitation by Northern blot analysis may require the use of an internal standard to normalize results caused by uneven RNA loading or transfer It is critical to first determine if the internal standard mRNA target is regulated by the paradigm being tested Several riboprobe templates are available, including actin and cyclophillin (Ambion [Austin, TX]) If the size of the Internal standard transcript is different from the target RNA, it is more efficient to hybridize both probes at once However, riboprobes and random prime labeled DNA probes cannot be used together, because they require different hybridization temperatures Therefore, If the target probe and the internal standard probe are not of the same type, they must be run separately It is important to remember that often the level of RNA encoding the commonly used internal standards far exceeds the level of the target RNA, especially if studying G protein-coupled receptors It is recommended that the internal standard probe be labeled to a lower specific activity than the tar&t probe 30 It is important to preheat the wash buffers to temperature prior to addition to the blot This will ensure that the appropriate wash conditions have been reached 1, The wash protocol outlmed here is just a starting point Additional or longer washes may be necessary 32 Before purifying the riboprobe, remove pL and determine counts/mm Column purify probe and determine counts/min Calculate percent mcorporatlon of radiolabeled nucleotide, and if it is less than approx 30%, discard probe In general, efficient riboprobe labeling reactions should incorporate 50-70% of the label A lower percent incorporation may indicate that the label has degraded, the template concentration is too low, and/or RNase contamination has been introduced into the labeling reaction 33 It is critical that the probe be added to samples so that it is m vast excess of the mRNA to which it is to hybridize Preliminary RPAs should be run on samples containing equal amounts of total RNA and increasmg amounts of probe, until a probe concentration is found that no longer yields an increased signal on an auto- 270 34 35 36 37 38 39 40 41 42 43 44 45 Iredale et al radiogram For most G protein-coupled receptors, x 105 counts/mm of high specific activity probe should be sufficient For end-labeling DNA size markers, the DNA must be digested with a restriction enzyme that leaves a 5’ overhang or blunt end; the correct radiolabeled nucleotide must be used Klenow fills in a 5’ overhang with a radiolabeled nucleotide that IS complementary to the overhang For example, a Hind111 digest leaves a 3’ TTAA 5’ overhang, and Klenow adds radiolabeled 5’ AA 3’ to the complementary strand For enzymes that leave a blunt end, Klenow replaces the nucleotide at the 3’ terminus of the restriction site For example, Hoe111 leaves a 5’ GG 3’ blunt end, and Klenow replaces the second G with a radiolabeled G When pouring gels using the Bio-Rad Mini-PROTEIN II gel casting stand, apply a thm film of petroleum Jelly to each silicone gasket to ensure that no leaking occurs Solutions for making polyacrylamide gels need not be DEPC-treated Expect 8% gels to run for approx 70-90 before the xylene cyan01 dye front reaches the bottom of the gel To prevent the apparatus from heating up, which could cause smiling of the sample fronts, precool the 1X TBE to 4°C before adding to the chamber 6% gels yield satisfactory results and are faster to run, but 8% gels yield nottceably sharper bands It is important not to premix 10% SDS with Proteinase K before adding them to RNase-treated samples, because such concentrated SDS may denature the protease DNA and RNA molecules of the same size electrophorese at different rates RNase-protected RNA fragments are approx 5-l 0% smaller m length than a DNA size marker traveling at the same rate The half-life will vary, depending on the particular species of interest; therefore, these times are merely a guideline Plot time (x-axis) agamst log % of control (v-axis) for both control and agonisttreated cells The half-life (50% of control) can be derived by extrapolatmg from the graph (most computer programs will perform this function Alternatively, the half-life can be determined from the slope of the line, using the formula: half-life = In 2/k, where In = 0.693 and k = -slope of the line (see ref IS) The lower the amount of DNA loaded per lane, the better the recovery Unfortunately, it is necessary to cut large quantities of plasmrd m order to obtain sufficient amounts of cDNA (at least 30 pg for SIX samples) to bmd the nascent RNA transcripts from the nuclear run-on assay Therefore, in order to keep the number of lanes to a practical number, it also becomes necessary to load larger amounts of cDNA per lane (20-40 pg of total DNA/lane) Handle the nylon with blunt forceps and gloved hands It is a good idea to have an internal standard that can be used to normalize the results This needs to be a gene that is ubiquitous and IS unaffected by the experimental conditions under observation, Cyclophilin and actin are commonly used examples; however, both can be affected by certain treatments Linearized vector (minus the cDNA of interest) serves as a useful second control Regulation of Receptor Expression 271 46 Use a colored pencil to mark the location on the membrane of the slots containing cDNA and also the orientation, of the blot, since it is very difficult to see anything after the membrane has dried 47 It is very important to use DEPC-treated water to make all the solutions, in order to minimize the possibility of RNase contamination 48 It is important to keep the nuclei free from RNase contamination DEPC-treat the Dounce homogenizers, pestles, and centrifuge tubes before use 49 Use a 5-mL transfer pipet to layer the homogenate Hold the centrifuge tube on a slight angle and drip the homogenate down the wall of the tube 50 The pellet can be quite hard to resuspend; however, it is necessary for later stages in the nuclear run-on assay that the nuclear suspension is as homogeneous as possible Therefore, take time to resuspend the pellet well by flicking the tube and pipeting the pellet up and down The nuclei will withstand a certain amount of stress and taking the time at this point will alleviate unnecessary operator stress later in the assay 51 Use 2-mL Eppendorf tubes with screw caps and rings to reduce radioactive contamination 52 The nuclei need to be passed through the needle at least three times to ensure complete shearing Considerable care should be taken during this procedure to avoid aerosolizing radioactivity 53 On average we try to add at least 12 x 1O6counts/mm/tube The higher the amount of radiolabeled RNA added, the better the hybridizations, assuming that the overall level of RNA synthesis is not changing as a function of cell state The assumption might not always be valid and should therefore be examined prior to embarking on new experimental conditions 54 Stop the second wash when the radioactivity is approx 200 counts/min (estimate using a Geiger counter) This is usually after 2-4 mm of the second wash; therefore, check the blots every to prevent over-washing Acknowledgments This work is supported by USPHS grants MH4548 1, MH53 199, and PO1 MH25642, and by a Veterans Administration National Center Grant for PTSD, VA Medical Center References Collins, S., Caron, M G., and Letkowitz, R J (1991) Regulation of adrenergic receptor responsiveness through regulation of receptor gene expression Annu Rev Physiol 53,491-508 Hadcock, J R and Malbon, C C (1991) Regulation of receptor expression by agonist: transcriptional and post-transcriptional controls Trends Neurosci 14, 242-247 Hadcock, J R., Wand, H., and Malbon, C C (1989) Agonist-induced destabrlization of P-adrenergic receptor mRNA J Biol Chem 264, 19,928-19,933 272 lredale et al Collins, S., Bouvier, M., Bolanowskr, M A., Caron, M G., and Lefkowitz, R J (1989) CAMP stimulates transcriptron of the P2-adrenergic receptor gene in response to short-term agonist exposure Proc Natl Acad Sci USA 86,4853-4857 Bahouth, S W (1991) Thyroid hormones transcriptronally regulate the pladrenergic receptor gene in cultured ventricular myocytes J Biol Chem 266, 15,863-15,869 Hosoda K and Duman R S (1993) Regulation of p 1-adrenergic receptor mRNA and hgand binding by antidepressants and norepinephrine depletron J Neurothem 60, 1335-1343 Butler, M O., Morinobu, S., and Duman, R S (1993) Chronic electroconvulsive seizures increase the expression of serotonin2 receptor mRNA m rat frontal cortex J Neurochem 61,1270-1276 Krely, J., Hadcock, J R., Bahouth, S W., and Malbon, C C (1994) Glucocorticoids down-regulate p 1-adrenergic-receptor expression by suppressing transcription of the receptor gene Biochem J 302,397-403 Hosoda, K., Feussner, G , Rydelek-Fitzgerald, L., Vidaya, V., Fishman, P H., and Duman, R S (1995) Regulation of P2-adrenergic receptor n-RNA and gene transcription in rat C6 glioma: effects of agonist, forskolin, and protein synthesis inhibition Mol Pharmacol 48,206-2 11 10 Port J D., Huang L -Y., and Malbon C C (1992) B-adrenergrc agonists that downregulate receptor mRNA up-regulate a Mr 35,000 protein(s) that selectively binds to b-adrenergic receptor mRNAs J Biol Chem 267,24,103-24,108 11 Sachs, A and Wahle, E (1993) Poly(A) tail metabolism and function in eucaryotes J Biol Chem 268,22,955-22,958 12 Beelman, C A and Parker, R (1995) Degradation of mRNA m eukaryotes Cell 81,179-183 13 Morgan, J and Curran, T (1991) Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun Annu Rev Neurosci 14,42 l-45 1, 14 Armstrong, R C and Montminy, M R (1993) Transsynaptic control of gene expression Annu Rev Neurosci 16, 17-29 15 Rodgers, J R., Johnson, M L., and Rosen, J M (1985) Measurement of mRNA concentration and mRNA half-life as a function of hormonal treatment Methods Enzymoi 109,572-592 ... bovine A, adenosme receptor and the rat A3 adenosine receptor to examine the structural requirements for ligand binding by adenosine receptors The two wild-type receptors, as well as receptor chimeras... binding can be identified; signal transduction pathways of a particular receptor can be delineated; interactions between a transfected receptor and other expressed receptors, both endogenous... Section 7.7 Chimeric Receptors The vast majority of chimeric receptors employed to study ligand binding are constructed from two parent receptors that belong to the same receptor family For example,