Glycoprotein Methods and Protocols - P3
Glycoprotein Methods and Protocols - P3
... presence of competitive inhibitors of O-glycosylation, such as benzyl 2-acetamido-2-deoxy-α-D-galactopyranoside, without adversely affecting cells (9). With the eluci-dation of more sequence data for ... specificity (nonmucin glycoproteins can react, no carbohydrate or core pro-tein specificity), and lower sensitivity than slot-blot and immunoassay methods. Chap-
Detection and Quantita...
Glycoprotein Methods and Protocols - P30
... supernatant and count the radioactivity.3.1.3. Glycoprotein Assay with β 1-3 Linked- and β 1-4 Linked-GalactoseRadioactive Substrates (seeNotes 9, 10, and 14)The detection of β 1-3 - and β 1-4 -specific ... asialo-ovinesubmandibular gland mucin, or α1-acid glycoprotein, in 500 µL of 50 mM sodium phos-phate and 5 mM NaCl, pH 7.0.2. Add 5 U of galactose oxidase (Sigma) to...
Glycoprotein Methods and Protocols - P31
... formate anion-exchange resin in 1-mL columns with taps.2.4.p-NitrophenylN-Acetyl-β-D-Glucosaminide-6-SulfateDesulfation Reagents1. Substrate: p-nitrophenyl N-acetyl-β-D-glucosaminide-6-sulfate (Industrial ... Product standard: p-nitrophenol (1.25 mM).2.5.p-Nitrophenyl-β-D-Galactoside-6-Sulfate Desulfation Reagents1. Substrate: p-nitrophenyl β-D-galactoside-6-sulfate (Industrial Research) (3.2...
Glycoprotein Methods and Protocols - P32
... (v/v).5. 3-kDa Cutoff centrifugal ultrafilters (Filtron, Northborough, MA).6. Precast, 4% and 4-2 0% Tris-glycine gels for SDS-PAGE (Novex, San Diego, CA).7. Kaleidoscope™ (Bio-Rad) and See-Blue ™ ... Precast, 4–20% Tris-HCl Ready Gel (Bio-Rad, Hercules, CA) for sodium dodecyl sul-fate-polyacrylamide gel electrophoresis (SDS-PAGE).6. Silver stain kit (Bio-Rad).7. Coomasie blue R-250 (Si...
Glycoprotein Methods and Protocols - P33
... solutions A and B at room temperature, and make solution C fresh daily.6. Make glycosidase assays using the following p-nitrophenyl substrates: N-acetyl α-D-galactosaminide, α-L-fucopyranoside, N-acetyl ... N-acetyl β-D-glucosaminide, and β-D-galacto-pyranoside, all prepared as 15 mM solutions in 0.01 M Tris buffer, pH 6.5. The stopsolution is a mixture of 0.5 M Na2CO 3and 0.5 M NaHCO3...
Glycoprotein Methods and Protocols - P34
... with L-cells transfected with DbcDNA, and specificity is confirmed in RMA-S cells.H2d is mapped similarly. For HLA-A*0201 epitope mapping, EBV-immortalized B-cells(HLA-A*0201, HLA-A1, HLA-A11, and ... form), glutathione-agarose, isopro-pyl-β-D-thiogalactopyranoside (IPTG), phytohemagglutinin (PHA), and ethanediol were fromSigma, St. Louis, MO). Sodium periodate, Triton X-100, and glut...
Glycoprotein Methods and Protocols - P35
... (1992) Antigen-specific, MHC-unrestricted T cells. Biotherapy 4, 239–249.4. Magarian-Blander, J., Ciborowski, P., Hsia, S., Watkins, S. C., and Finn, O. J. (1998) Inter-cellular and intracellular ... underglycosylated MUC1 mol-ecule on tumor cells can be recognized as a tumor-specific antigen by T-cells. Themajor cytotoxic T-lymphocyte (CTL) response to tumor-specific MUC1 is T-cellrecept...
Glycoprotein Methods and Protocols - P36
... recombinant transfer plas-mid pIE 1-4 -TR–-MUC1; 400 ng of pIE-neo plasmid DNA, and 20, 40, 60, 80, or 100 µLof Insectin-Plus liposomes. For mocktransfection, use 20 µL of Insectin-Plus liposomes.4. ... onEpstein-Barr Virus-immortalized B-cells and Burkitt’s Lymphomas transfected with epi-thelial mucin complimentary DNA. Cancer Res. 52, 5985–5990.4. Magarian-Blander, J., Domenech, N.,...
Glycoprotein Methods and Protocols - P37
... RPMI-1640 HEPES-buffered medium plus 336 mLof 3.3% trisodium citrate in distilled water and 14 mL 5 µM β-mercaptoethanol. Ali-quot 25 mL of this into sterile 50-mL conical screw-capped tubes and ... adher-ent cells in interleukin-4 (IL-4) and GM-CSF (12,13), whereas mouse dendritic cellscan be obtained from bone marrow cells by culturing in the presence of granulocyte-macrophage colon-s...
Glycoprotein Methods and Protocols - P38
... Baeckström, D., and Hansson, G. C. (1996) Distinct sub-populationsof carcinoma-associated MUC1 mucins as detected by the monoclonal antibody 9H8 andantibodies against the sialyl-Lewis a and sialyl-Lewis ... tandem repeats (TRs) of the MUC1peptide core, i.e., NH 2-( HGVTSAPDTRPAPGSTAPPA)3-COOH. The 60 mer peptide isconjugated to BSA (Sigma, St. Louis, MO, RIA grade, cat. no. A-7888) usin...