A 25ml Sera
24-GST-Pip1-G (day 84)
24-MBP-Pcu1-C (day 77)
24-MBP-Skp1-E (day 84)

• thaw sera in a waterbath
• fill up to 25ml with PBS
• spin in glass corex/transpartent plastic tubes, 15k, 10min, 4° C (SS-34); balance with PBS
• collect supernatant and discard pellet
• add (slowly) 4.4g Ammoniumsulfate (30%) to supernatant (25ml), use a stirrer
• spin in glass corex/transpartent plastic tubes, 17k, 20min, 4° C

• discard pellet
• add (slowly) 3.1g Ammoniumsulfate (50%) to the supernatant
• spin in glass corex/transpartent plastic tubes, 17k, 20min, 4° C
• resuspend pellet in 10ml PBS, discard supernatant
• spin in glass corex/transpartent plastic tubes, 17k, 20min, 4° C
• dialyze supernatant o/n in PBS

Do not use buffers such as Tris or glycine.

The Affi-gel 10 support couples proteins best at a pH near or below their isoelectric point, and the Affi-Gel 15 support couples proteins best near or above their isoelectric point. Therefore the Affi-Gel 15 support is recommended for proteins with isoelectric points below 6.5 (acidic proteins).

• Dialyze proteins o/n against 100mM Sodium-bicarbonate buffer (» pH8.2)

MBP-Pip1 (» 5.5 ml; 2.8 mg/ml)
MBP-Pcu1 (» 2.8 ml; 1 mg/ml)
GST-Skp1 (» 2.5ml; 4 mg/ml)

• Affi-Gel 15: shake vial and make sure that you have a uniform suspension
• transfer 2ml of a 50% slurry to a column or glass fritted funnel
• drain the supernatant solvent
• wash the gel with three bed volumes of water

for optimum coupling the washing procedure should be completed and the gel combined with the ligand solution within 20min

• add » 1ml beads to protein

add at least 0.5ml of ligand solution per ml of gel
protein concentration of <25mg/ml of gel will yield less than optimum result
volume of ligand should not exceed 4.5ml/ml of gel
• incubate o/n (or at least 4h) at 4° C and agitate sufficiently to make a uniform suspension
• test supernatant and beads with Bradford reagent

compare to input solution
• add 0.1ml/ml gel of a 1M Ethanolamine-HCl, pH 8 solution
• block for 1h
• let gel settle down for about 10min
• remove supernatant and store at 4° C (for security reason)
• resuspend gel in 5ml PBS
• transfer the gel to a column
• wash with 10ml PBS (pump speed » 1ml/min [» level 7])

• add beads (coupled with protein) to column (see B)

for all next steps: pump speed » 1ml/min (» level 7)
• wash with 10ml 100mM Glycine-HCl pH 2.5
• wash with 10ml 10mM Tris-HCl pH 9
• wash with 10ml 100mM Triethanolamine pH 11.5
• wash with 10ml 10mM Tris-HCl pH 6.8
• wash with 10ml PBS

• load antibodies to column (see A)
• 2 h circuit
• wash with 20ml 10mM Tris-HCl pH 7.5

one can increase pump speed from level 7 to level 9
• wash with 20ml 10mM Tris-HCl pH 7.5, 500mM NaCl

• wash with 20ml 10mM Tris-HCl pH 7.5

Next steps: it is important to remove the eluted antibody from the eluant as quickly as possible to minimize the chance of denaturation. The sample should be neutralized immediately following elution!

• elute with 100mM Glycine-HCl pH 2.5: 1ml into 100m l 1M Tris-HCl, pH 9 (about 10 tubes)

mix tubes carefully
check protein concentration with Bradford
pool positive fractions
• neutralize column with 10mM Tris-HCl pH 7.5 (about 10ml)
• check pH
• elute with 10ml 100mM Triethanolamine pH 11.5: 1ml into 100m l 1M Tris-HCl, pH 6.8 (about 10 tubes)

mix tubes carefully
check protein concentration with Bradford
pool positive fractions
• neutralize column with 10ml 10mM Tris-HCl pH 7.5 (about 10ml)
• wash column with 10ml PBS, 1mM DTT and store at 4° C
• concentrate pooled antibody fractions with 10k concentrators (ca. 250mg /ml )

block concentrators before use with 3% BSA
• add Thimerosal (1x final, =0.1%)
• store at 4° C
• test antibodies