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4. Wash the column with PBS until no more protein elutes from the column, using a spectrophotometer or UV monitor (OD 260) to confirm the end-point. 36 5. 6. 7. 8. , 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.

14) with 5 µg of ovalbumin conjugated to 1 mg of alum. Sera or plasma from these mice can be used as reference standards. 16 In our hands, the streptavidin-horse radish peroxidase/ ABTS enzyme/substrate combination gives vastly superior results to those obtained with the SA-AP/p-nitrophenyl phosphate enzyme/substrate system although, in all honesty, we have not "played" with the latter system sufficiently to suggest that it could not yield equivalent results under the correct circumstances. 58 5.

High concentrations of ammonium sulfate will cause the proteins in a solution to differentially precipitate out from their solubilized state - at 30% ammonium sulfate saturation, most of the IgM antibodies will precipitate out of solution, while at 45% saturation most of the IgG isotypes and the residual IgM antibodies will precipitate out of solution. 45 µm filters pH meter and pH reagents stir plate syringe and 20 ga needle Reagents culture medium from a terminal culture of TIB211 (IgM anti-CD8) hybridoma cells saturated ammonium sulfate solution borate-buffered saline Method 1.

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