Protein G Agarose IP Reagent

Protein G is provided as an agarose conjugate for use in immunoprecipitation only. The product is provided as 0.5 ml agarose in 2.0 ml PBS. Protein G-Agarose is pre-blocked with BSA to reduce non-specific immunoglobulin binding. Sufficient product is provided for 100 immunoprecipitation reactions, to be used at 20 ul resuspended volume per reaction.

1. Incubate cultured cells (80 - 90 % confluent monolayer in 100 mm cell culture plate, or approximately 2 - 5 x 107 suspension cells in flask) in methionine-free medium containing 5 % dialyzed fetal calf serum for 1 hour at 37 °C. The same procedure can be used for cells labeled with other radioactive amino acids (e.g., 14C or 3H) or with γ32P-orthophosphate. Cell labeling must be carried out in medium lacking the relevant amino acid or in phosphate-free medium.
2. Remove medium and replace with 3 mLmethionine-free medium containing 5 % dialyzed fetal calf serum and 100 uCi/mL 35S-methionine. Incubate 1 hour at 37 °C. For some proteins a longer labeling period (up to 18 hours) is preferable.
3. Carefully remove radioactive medium with Pasteur pipette and wash cell monolayer with PBS.
4. Add 3 mLice cold RIPA buffer to cell monolayer and incubate at 4 °C for 10 minutes. For suspension cells, add the RIPA buffer to washed cell pellet in a 15 mLconical centrifuge tube.
5. Disrupt cells by repeated aspiration through a 21 gauge needle and transfer to a 15 mLconical centrifuge tube.
6. Wash cell culture plate with additional 1.0 mLice cold RIPA buffer and combine with original extract.
7. Pellet cellular debris by centrifugation at 10,000xg for 10 minutes at 4 °C. Transfer supernatant to a fresh 15 mLconical centrifuge tube on ice. Preclear lysate (optional step) by adding 1.0 μg of the appropriate control IgG (normal mouse, rat, rabbit or goat IgG, corresponding to the host species of the primary antibody), together with 20 μL of resuspended volume of Protein G-Agarose. Incubate at 4 °C for 30 minutes.
8. Pellet beads by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4 °C. Transfer supernatant (cell lysate) to a fresh 15 mLconical centrifuge tube on ice.
9. Transfer 1 mLof the above cell lysate, or approximately 100 - 500 μg total cellular protein, to a 1.5 mLmicrocentrifuge tube. Add 1 - 10 μL (i.e., 0.2 - 2 μg) primary antibody (optimal antibody concentration should be determined by titration) and incubate for 1 hour at 4 °C.
10. Add 20 μL of resuspended volume of Protein G-Agarose. Cap tubes and incubate at 4° C on a rocker platform or rotating device for 1 hour to overnight.
11. Collect immunoprecipitates by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4 °C. Carefully aspirate and discard radioactive supernatant.
12. Wash pellet 4 times with 1.0 mLRIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above.
13. After final wash, aspirate and discard supernatant and resuspend pellet in 40 μL of 1x electrophoresis sample buffer.
14. Boil samples for 2 - 3 minutes and analyze 20 μL aliquots by SDS-PAGE and autoradiography. Unused samples may be stored at -20 °C.
15. Optional: After boiling, samples may be centrifuged to pellet the agarose beads followed by SDS-PAGE analysis of the supernatant.