1. Blot proteins onto nitrocellulose by standard electrophoretic transfer, visualize the proteins by Ponceau S staining, and cut out the band of interest. To purify Abs from 2 ml of high titer serum, want to load about 1 mg of your protein on a 1.5 mm thick, 16 cm wide gel. Want to run as much protein as will run without streaking on this gel. If the sample is of high enough purity it can be loaded on only half of such a gel. The amount of protein you can load without streaking depends on the purity of your sample, and will have to determined empirically. The large band that you eventually cut out of the blot will be about 7-30 cm2, depending on how much of the width of the gel you loaded and how well the gel ran. I let the destained membrane strip dry to ensure tight binding of the protein.
1a. Alternatively, apply proteins to dry membrane, at ~0.1 mg/cm2.
2. Remove poorly bound protein by treatment with 100 mM glycine/HCl pH 2.5 for 5 min. Wash membrane 2X 2 min. in TBS.
3. Block membrane with 3% BSA ("Pentax Fraction V") in TBS for 1 hour at room temp, on a rocker. Wash 2X 2 min in TBS.
4. Dilute 2 ml serum with 8 ml TBS and allow it to bind to membrane 2-3 hour at room temperature, or overnight at 4°. This can be done easily by cutting the membrane into small strips (~1 cm X 0.5 cm), stuffing them into a Falcon 2059 tube (12 ml snap cap), adding liquid, and incubating on a rocker.
5. Save the supernatant, and wash the filter 2X 5 min. with TBS.
6. Wash 2X 5 min. with PBS.
7. To elute: add 1 ml glycine, incubate 10 minutes with occasional vortexing.
8. Remove glycine to a tube containing a volume of 1 M Tris pH 8.0 that will bring the final pH to 7.0. This should be about 100 µl Tris for 1 ml glycine.
9. Repeat the elution step. Store membranes in PBS with azide. They can be reused a few times.
10. Pool eluates. There is some white precipitate in the mix (I don't know if this is protein or nitrocellulose bits), so spin these out in a microfuge. Add sodium azide to 5 mM and BSA to 1 mg/ml to stabilize the purified antibody.
11. Store aliquots at -80° for long term storage; can keep an aliquot for current use at 4° for several months.
12. To check the purification, do a series of semiquantitative test Westerns to determine the yield of purified antibody. This will also titer your purified material.
A. When using bacterial recombinant protein, prepare two gel samples: 1) positive control - whole cell lysate of E. coli expressing the recombinant protein; and 2) negative control, whole cell lysate of E. coli not expressing the protein. Run a gel on which these samples are run every other lane, blot the gel, and cut the blot into strips each having a postitive and negative control lane. You should load only a very small amount of extract per lane: I recommend 0.0125 OD ml (an OD ml is the amount of E. coli in 1 ml of culture at OD550=1.0).
B. Probe the test strips with several dilutions of the starting material, the supernatant from step 5, and the purified material. You must use a low-sensitivity development method in order to be able to compare the intensity of the signals on the various blots (ECL is inappropriate: the signal will be saturated too easily). I recommend using an alkaline phosphatase-conjugated secondary antibody and developing with BCIP and NBT to get purple bands on the blot. Make sure you develop all the blots simunltaneously in one container of developer for the same amount of time so that you can compare the signals on all the blots.
C. Typically, I absorb 100% of the specific antibody from 2 mls of serum, and recover about 20% of the Western activity in the purified material.
TBS
20 mM Tris pH 7.4
500 mM NaCl
0.05% Tween-20
PBS
20 mM sodium phosphate pH 7.2
150 mM NaCl