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Purification of Rabbit Antiserum With Agarose Protein-A beads
Material and Reagent
- Agarose beads, Bio-Gel A supports, Cat: 151-0150, Source: BIO-RAD
- Agarose protein A, Vector laboratories, SP-0050
- Tris-HCl buffer, 1.0M PH8.0
- Tris-HCl buffer, 100mM PH8.0
- Tris-HCl buffer, 10mM PH8.0
- glycine solution 100mM adjusted to PH3.0 with acetic acid
Proceedure
- Wash the agarose[ Bio-Gel A supports, 151-0150] with 0.01M phosphate buffered saline (0.15M NaCl, PB: PH7.5) 3 times X 10 minutes
- Wash the agarose protein A [ Vector laboratories, SP-0050] with 0.01M phosphate buffered saline (0.15M NaCl, PB: PH7.5) 3 times X 10 minutes
- Take 1 ml antiserum, and add 100 ml Tris-HCl buffer 1.0M, PH8.0
- Mix the adjusted antiserum with washed agarose on a rocking plate for 10 minutes
- Centrifuge for 5 minutes at 1000RPM
- Collect the supernatant
- Put the supernatant into washed protein A agarose, mix with gentle shaking for 1 hour at 4° C
- Centrifuge for 5 minutes at 1000RPM
- Discard the supernatant
- Wash with Tris-HCl buffer 100mM, PH8.0
- Centrifuge for 5 minutes at 1000RPM
- Discard the supernatant
- Wash with Tris-HCl buffer 10mM, PH8.0
- Discard the supernatant
- Elute the antibody with 500ml 100mM glycine acetic buffer PH3.0, shaking on a rocking plate for 10 minutes
- Centrifuge for 5 minutes at 1000RPM
- Collect the supernatant and add 50 ml of 1.0M Tris-HCl buffer, PH8.0 (1/10 volume of elutate buffer)
- Put 1 volume of glycerol to the purified antibody, and aliquat and store in -80° C
- Wash the beads with phosphate buffered saline 0.15M NaCl, PB: PH7.5
- Store the beads at 4° C. It can be reused for purification the same antiserum.