Protein A Separopore® 4B Column
Protein A Separopore® 4B
Affinity matrix with immobilized Protein-A (from staphylococcus aureus) by CNBr activation coupling method. For purification and/or fractionation of IgG subclasses. Reliable tool to purify monoclonal and polyclonal IgG from ascies, serum and cell culture supernatants.
Provided as conveniently prepacked, ready-to-use spin columns for gravity flow chromatography.
Note: Separopore® is a cost-effective equivalent to Sepharose® in all of its physical properties and binding characteristics.
IP with Protein A
• Add 3 ml ice cold RIPA buffer to cell monolayer (or approximately 2-5 x 107 suspension cells in flask) and incubate at 4º C for 10 minutes. For suspension cells, add the RIPA buffer to washed cell pellet in a 15 ml conical centrifuge tube.
• Disrupt cells by repeated aspiration through a 21 gauge needle and transfer to a 15 ml conical centrifuge tube.
• Wash cell culture plate with additional 1.0 ml ice cold RIPA buffer and combine with original extract.
• Pellet cellular debris by centrifugation at 10,000xg for 10 minutes at 4º C. Transfer supernatant to a 15 ml conical centrifuge tube kept 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)towns add 20 µl of resuspended volume of Protein A-Separopore. Incubate at 4º C for 30 minutes.
• Pellet beads by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4º C. Transfer supernatant to a fresh 15 ml conical centrifuge tube kept on ice.
• Transfer 1 ml of the above cell lysate, or approximately 100-500 µg total cellular protein, to a 1.5 ml microcentrifuge 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 4º C. longer incubation. May be. Required for some low titter antibodies.
• Add 20 µl of resuspended volume of Protein A-Separopore. Cap tubes and incubate at 4º C on a rocker platform or rotating device for 1 hour.
• Collect immunoprecipitates by centrifugation at 2,500 rpm (approximately 1,000xg) for 5 minutes at 4º C. Carefully aspirate and discard supernatant.
• Wash pellet 4 times with 1.0 ml RIPA buffer (more stringent) or PBS (less stringent), each time repeating centrifugation step above.
• After final wash, aspirate and discard supernatant and resuspend pellet in 40 µl of 1x electrophoresis sample buffer.
• Boil samples for 2minutes and analyze 20 µl aliquots by SDS-PAGE and follow it by Western blot analysis or auto radiography if sample was labeled.