By John R. Gordon
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Additional resources for A Practical Guide to Cellular and Molecular Research Methods in Immunology, 2nd Edition
Bio-Rad, Bradford or CBB] protein assay; Appendix D), and pool the protein-containing fractions. Regenerate the column with ≈10 ml of PBS, and store the matrix in PBS/20% ethanol. ). After dialysis, determine the protein concentration of the eluted IgG solution using a and, if necessary, concentrate the eluted protein using a centrifugal concentrator. , protein A, T gel, Avid-Chrom), so alternate methods must be employed to prepare IgM antibodies. A number of methods are available, with the simplest true purification probably being achieved by size exclusion chromatography (IgM pentamers have a molecular mass of ≈750 kD, while IgG and albumin have molecular masses of 150 & 65 kD, respectively).
METHOD 7 Expression of the FcεRI is inducible in mast cells, and this is at least in part regulated the concentrations of exogenous IgE, such that in the presence of high concentrations of IgE, mast cells express more IgE receptors. Thus, freshly purified tissue mast cells, which will likely have the vast majority of their existing FcεRI occupied by IgE antibodies of irrelevant specificities, can be best sensitized with IgE of the desired specificity by incubating the cells overnight in high concentrations of the antibody.
6. Overlay the tissue sections with ≈75 µl of the biotinylated secondary antibody, again using empirically-determined optimal concentrations of the antibodies, generally for 2h @ RT. 7. Wash the tissue sections three times for 5' each in PBST. 8. Overlay the tissue sections again, this time with SA-AP diluted to 1:5000 in PBST, and incubate for 90 ' @ RT to label the secondary antibodies in the tissue sections. 9. Wash the tissue sections three times for 5' each in PBST. 10. Overlay the tissue sections with the commercial solution of BCIP/NBT for 20 - 40' @ RT (continue staining until the antigen of interest becomes apparent or until a non-specific general tissue background staining begins to appear).