Affinity purifying antibodies with protein-antigens immobilized on nitrocellulose.

Immobilizing the antigen on nitrocellulose.

1. Run 100 ug of purified protein on SDS-PAGE (15% SDS polyacrylamide gels work well for small recombinant proteins (10 to 30 kD)) using the Miniprotein gel apparatus from BioRad.  The stacking gel is made so it provides one large well almost spanning the full width of the gel.
   
• These proteins are often purified on a nickle column and are in urea buffer.  Individual components of SDS-PAGE buffer are added to give a final volume of 300 ul.  Samples are boiled for 5 minutes before loading.
• The amount of protein could easily be increase to 200 ug without overloading the gel.
• RD (30kD) ran about 1/3 of the way down the gel while C/D (14 kD) ran a little past half way.
• The samples are loaded in one wide slot.
• The gel is run at 50 volts until the sample has traversed the stacking gel, and then the voltage is increased to 100.
2. The gel is blotted to nitrocellulose using the dry-blot apparatus.  The blotting is done for 1 hour at 10 volts.
3. Filters are stained with Poncue Red according to the antibody book and destained briefly with PBS.  The desired protein should appear as a red line, about 2mm in thickness.
4. The red line of protein was excised with a razor blade and cut into 0.5 cm long chips.  The chips were put in 1 ml of PBS and stored in the refrigerator.  As a negative control, you can prepare comparably sized chips with a different protein or with no protein.
   

Affinity purifying the antibody.

1. In a 500 ul tube, incubate a nitrocellulose chip for 1 hr at 4^oC in 100 ul of 10%FBS (diluted in TBS).  If you are doing this for the first time, its a good idea to do a mock antibody purification using a nitrocellulose chip that lacks protein or contains a protein that doesn't bind your protein of interest.
   
2. Without removing the 10% FBS solution, add 100 ul of crude antiserum to the tube.  Mount on a rotating wheel and mix overnight at 4^oC.
3. Carefully pipette away the solution and wash the chip 4 times with 400 ul of TBS.  Each wash should be for 10' at 4^oC.
4. Using a syringe needle, make a small hole at the bottom of the tube containing the chip and remove the cap.  Place the tube in a clean 1.5 ml tube and briefly centrifuge so moisture on the chip is spun out the bottom of the tube.
5. Place the tube with the chip on its side and move the chip so that it is located approximately half-way up the side.  Overlay the chip with 30 ul of 100 mM glycine pH 2.5 and incubate at room temperature for 10'.
6. Put 5 ul of 1M Tris pH 8.0 and 2.5 ul of 100% FBS in the bottom of a 1.5 ml tube.  Remove and save the cap.  Place the 500 ul tube containing the chip into the 1.5 ml tube and briefly spin the glycine solution through the hole at the bottom of the 500 ul tube into the solution at the bottom of the 1.5 ml tube.
7. Again, place the 500 ul tube on its side, move the chip half-way up the side, and overlay the chip with 15 ul of 100 mM glycine pH 2.5.  Incubate for 10' at room temperature.
8. Place the 500 ul tube into the 1.5 ml tube and spin the second elute into the first elute.
9. Store affinity purified antibody at 4^oC.  Save the chip in PBS for possible reuse.

Immunofluorescence.

1. Test the purified antibody at 1/10 dilution.  It is possible that a greater dilution will work but this is a good dilution to start with.