E coli Shiga Toxin Methods and Protocols doc

... loading buffer was added to 15 m L of each subfragment PCR product and the samples were mixed well. 3. The samples were loaded onto the agarose gel submerged in TAE buffer and electrophoresed at ... subfragment were screened for correct insert size by EcoRI digestion and the 5 ¢ Agg inserts for correct orientation by XbaI digestion. Positive candidates were sequenced to confi rm in...

Ngày tải lên: 05/03/2014, 23:20

... the interface between the external and the internal environment of the organism, and represent the fi rst line of defence of our body. These genes often have an extensive region of repetitive ... boxes represent sequences missing in the human genome sequence. For MUC5AC , the complete genomic sequence is not known; therefore, the sequence and size of the intron from exon 15 to the ta...

Ngày tải lên: 14/03/2014, 22:20

... be pierced with a needle to equilibrate the pressure, and the DFP should be transferred using a syringe and needle. The contents of the vial should be placed directly into the correct volume ... adhere to the membrane may be lost and the linear range of the technique may be limited. “Coating methods such as ELISA are prone to artefacts if samples are concentrated, and care must be ta...

Ngày tải lên: 08/10/2012, 10:25

... site has been selected for study, it is desirable that the results be presented in a standardized manner. The size of the area to be assessed may be predetermined, but this is more likely to be ... blood vessels. Whereas at least some positive structures are not guar- anteed in every section, sections from positive tissue that been fixed and processed in the same manner as the test samples...

Ngày tải lên: 08/10/2012, 10:25

... although effective against the protein precursors, were of little use for mature mucins. Nevertheless, chemical and/ or enzymatic deglycosylation techniques can be used to increase the effectiveness ... homology and therefore cross- reactivity (5). Many of the early MUC-specific probes were generated against sequences underlying the highly glycosylated tandem repeat regions of the molecule...

Ngày tải lên: 08/10/2012, 10:25

... adherent to the mucosal surface in vivo is determined by a balance between the rate of secretion by the underlying epithelium and the rate of erosion by mechanical shear associated with the digestive ... adherent mucous thickness in situ were based on (1) differences in refractive index between the mucous layer and the mucosal surface, measured by a slit lamp and pachymeter (16); (2) th...

Ngày tải lên: 08/10/2012, 10:25

... tissue sample. These parameters have been determined for a wide variety of gastrointestinal tissues and cell lines (e. g., see refs. 4–10), and are described in detail in Chapter 19. 9. The procedures ... to be able to measure mucin synthesis in a specific manner, i .e. , to determine the level of gene- specific expression of mucin. This can be done at the mucin mRNA level, as described e...

Ngày tải lên: 08/10/2012, 10:25

... alkylated in our analyses) that are characteristic of this molecule (3). These peptides arise from cysteine-rich regions that are repeated several times in the MUC5B polypeptide (7). 8. We have used ... above the gel surface and always use the same buffer volume. 3.3.2.4. W ESTERN B LOTTING After electrophoresis, transfer the gel to nitrocellulose or poly(vinylidine difluoride) (PVDF) (see...

Ngày tải lên: 08/10/2012, 10:25

... scattering and analytical centrifugation are not described here because they require expensive, specialized equipment and a detailed knowledge of their theoretical basis. 2. Materials 2.1. Extraction ... rotor geom- etries) isokinetic sedimentation behavior in this system (see Note 5). Heterogeneity and Size Distribution of Gel-Forming Mucins 89 We next describe an experiment that invol...

Ngày tải lên: 08/10/2012, 10:25

... been extensively vali- dated (1,2). Although the detailed protocols on the use of the automated GBC AminoMate (GBC Scientific) amino acid analyzer have been described elsewhere (3), here, we emphasize ... C18 reversed-phase column and detected by fluorescence. Our routine analyses have shown that the method described here is reliable and useful for both quantitative and qualitative mu...

Ngày tải lên: 08/10/2012, 10:25