ANTIBODY PURIFICATION by affinity chromatography
1) To affinity purify antibodies, generate lots of E. coli lysate that contains your antigen. If the protein can stand freeze thawing, then go ahead and purify the protein from e. coli lysate and keep it frozen until you need to couple it to a CH-sepharose column.
2) Using your purified antigen, weigh out some CH-separose 4B. Use about 1gram since the coupling capacity of the resin is around 1mg to 3mg/mL of swollen resin.
3) See protocol “Activated CH-sepharose” for coupling protein to CH-sepharose.
4) Meanwhile, Spin antisera/bleed at 32rpm 1 hour at 4°C in the ultra. Write down or take the sticker off the bottle and put it in your notebook with this purification since sometime it may be a bad bleed and you need to go back and toss all of that batch. (Instead of the 32rpm spin, some people add an equal volume of saturated 4.1M ammonium sulfate, pH7.0 dropwise to the stirring antisera, they stir 1 hour and let it sit O/N at 4°C and centrifuge at 5rpm the next day in the sorvall to do an ammonium sulfate cut. The pellet is firm like gelatin or agar, decant supe, add a few mLs of PBS to wash the pellet and tube lightly. Chop up the pellet with a spatula and dissolve the pellet in 1/4 of the original volumeof PBS, dialyze the resuspended pellet in PBS to get rid of ammonium sulfate with 4 changes for 45 minutes at RT).
5) Load supernatant onto antigen-coupled Ch-sepharose4Bbeads in a small yellow Biorad column. Make sure that you collect the flow through and save it. Save it, since often, perfectly good extra antibodies flow through- use a dilution of the FT to test on strips to see if there is any there.
6) Wash the column with 20 column volumes of 1X PBS.
7) Wash the column with 20 column volumes Li wash (1M LiCl, 150mM NaCl, 0.5% Nonidet P-40 a.k.a. Igepal, 10mM Tris, pH 8.0).
8) 200mls PBS wash
9) Elute the column with 100mM Triethanolamine in cold water* (Molarity of this is 7.2134M, Sigma T0886, MW=101.2). Do 6x10mL eluates, and elute them directly into a falcon tube containing 1mL 10X PBS and 1% BSA, add around 350uL 2N HCL and check that the pH is 7.0 before any prolonged amount of time. BSA is added for freezing and stability.
10) Check all of the elution fractions and flowthrough on blot strips to find out which fractions the antibody is in or if it is still coming off of the column. Once you have checked on strips (start with a 1:50 dilution) then you can concentrate the antibodies down using solid sucrose dialysis. Aliquot the antibody into appropriate volumes in eppendorfs (about 50 to 100uL) and place them in the –80°C antibody freezer rack.
* you can also elute the antibodies with a low pH solution like 200mM glycine, pH 2.0, neutralize the pH with phosphate buffer.
***In a pinch, you can run out a bunch of antigen (around 1mg) on a gel, blot it and incubate a small amount of bleed with the cut out portion of your protein and wash it like above and elute in a small volume of glycine or triethanolamine and neutralize the pH.
** You can also try to get rid of crossreacting bands by doing the opposite- incubating the antibody bleed with every part of the lysate on a blot except for the antigen.