By John R. Gordon
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Extra resources for A Practical Guide to Cellular and Molecular Research Methods in Immunology, 2nd Edition
5 h at room temperature. , flicking and banging on paper towels). Careful washing is important, for it will reduce the assay background substantially. 9. Add 100 µl of freshly prepared BCIP/NBT substrate to each well. 37 µl NBT). 10. Incubate the plates at room temperature in the dark until spots develop or the plate background begins to increase (approximately 30-45 minutes). 55 11. To stop the reaction, flick out the substrate and wash the plates 3 times in H2O by the dunking method. Allow them to dry overnight (with the lids off), as the background fades dramatically when the plates dry.
Add 100 µl of the ABTS substrate (SAHRP avidin-enzyme conjugate only) or freshly prepared 3 mMp-nitrophenyl 7. , a drawer works well) and allow the reactions to develop for 20 - 45 min 17 at room temperature. The plates can be read directly at this point, or stop solution may be added to reduce plate to plate variability when reading multiple plates. For the ABTS substrate, the stop the reactions by adding 100 µl of 1% sodium dodecyl sulfate (SDS) to each well. Read the plates using the ELISA plate reader, set at a reading wavelength of 405 nm.
1 ml/tube). 0). a. 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).
A Practical Guide to Cellular and Molecular Research Methods in Immunology, 2nd Edition by John R. Gordon