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 sp-0050 or GIBCO 15918-014
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 with acetic acid
Procedure
- Wash the agarose and agarose protein A separately with 0,01M phosphate buffered saline (0.15M NaCL,PB: pH7.5)
Use 1ml agarose or agarose proteinA for 1ml serum.
Add 9-10ml PBS to 1ml agarose or agarose protein A
Mix, Centrifuge at 2000RPM 4°C for 10min X3. - Take 1ml antiserum and add 100ul Tris-HCL buffer 1.)M pH8.0.
- Mix the adjusted antiserum withwashed agarose on arocking plate for 10min.
- Centrifuge for 5 min at 2900RPM.
- Collect the supernatant
- Put the supernatant into washed protein A agarose, mix with gentle shaking for 1hour at 4°C.
- Centrifuge for 5 min at 2000 RPM.
- Discard the supernatant
- Wash with Tris-HCL buffer 100mM, Ph8.0
- Centrifuge for 5 min at 2000 RPM
- Repeat from8-10.
- Discard the supernatant
- Wash with Tris-HCL buffer 10mM, Ph8.0
- Centrifuge for 5 min at 2000 RPM
- Repeat from 12-14.
- Discard the supernatant.
- Elute the antibody witrh 500ul 100mM glycine acetic buffer pH3.0, Shaking on a roking plate for 10min.
- Centrifuge foe 5 min at 2800 RPM.
- Collect the supernatant and add 50ul of 1.0M Tris-HCL buffer, pH8.0(1/10 volume of elution buffer)
- Put 1 volume of glycerol to the purified antibody, aliquot and store in -20°C or -80°C.
- Wash the beads with PBS (0.15M NaCL,PB: pH7.5)
- Store the beads at 4°C. It can be reused for purification the same antiserum,
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