Fab preparation

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Buffers Required:

Note: THe volume of buffers prepared is dependent on the starting volume of antibody-containing media.  Adjust volumes accordingly.

To 800ml ddH2O add:

  • 8 g NaCl
  • 0.2 g KCl
  • 1.44 g Na2HPO4 (sodium phosphate dibasic)
  • 0.24 g KH2PO4 (monopotassium phosphate)

pH to 7.4, and bring up to 1L with ddH2O. Autoclave to sterilize.

Saturated Ammonium Sulphate (SAS):

To 1000 ml of double-distilled water add:

Dissolve at 50C to 60C.  Stand overnight at room temperature, then adjust pH to  6.8-7.0 with 5 or 10 M NaOH. Measure the pH using small aliquots.

Digestion Buffer:

Per 100 ml of water add:

  • 355 mg (20mM) sodium phosphate dibasic
  • 292 mg (10mM) EDTA

pH to 7.0.  Immediately before use add 20mM cysteine-HCl

10mM Tris-HCl

To 100ml of water add

  • 158 mg Tris-HCl

pH to 7.5.

IgG Fractionation

Based on [4]

  1. Dilute antibody 1:2 with PBS on ice.  E.G. 10ml of hybridoma media would be diluted with 20ml of PBS.
  2. With gentle stirring, on-ice, add SAS drop-wise to reach a final concentration of 45% ammonium hydroxide (see calculator below).  Warning: stir gently.  Avoid foaming, as this indicates protein denaturation.
  3. Once SAS addition is complete stir for 30min on-ice, then centrifuge at 1000g for 15 min at 4°C.
  4. Wash the precipitate with 45% SAS, then re-pellet at 1000g for 15 min at 4°C.
  5. Redissolve the precipitate in the same volume of PBS as the original antibody solution.
  6. Centrifuge at 5000g for 15 min at 4°C to remove any particulates.
  7. Transfer the supernatant to a clean tube and reprecipitate immunoglobulin using 40% SAS
  8. Centrifuge at 1000g for 15 min at 4°C.
  9. Redissolve the precipitate in a minimum volume of digestion buffer without cysteine (0.5 mL) and dialyse against 5L of digestion buffer without cysteine at 4°C overnight.
  10. Centrifuge at 5000g for 15 min at 4°C to remove more insoluble material.
  11. Spec the protein to determine the concentration.
SAS Volume Calculator:

For 1ml of antibody solution add:

File:SAS Calc.gif, where

SF = final saturation, Si = initial saturation.  Example, for 45% of a 1ml starting volume with no prior SAS addition:

Volume (ml) = 1(0.45-0) / (1-0.45) = 0.82ml

Fab Preparation

  1. Add 20mM cystein-HCl to the fractionated IgG & digest buffer.
  2. Dilute fractionated IgG to a concentration of 10mg/ml in digest buffer.
  3. Mix immobilized papain beads (Thermo #20341) to obtain an even suspension.  Take 500ul of slurry* for every 1ml of diluted IgG to be processed.  Transfer slurry to a clean tube large enough to perform the digest in.
  4. Wash gel 2X in 4ml/500ul of slurry with digest buffer.  Centrifuge briefly (1-2 min at 250 x g) to pellet papain.
  5. Pellet washed papain, and resuspend in 0.5ml of digest buffer per 500ul of starting slurry.
  6. Add antibody solution to papain slurry.
  7. For non-human IgG, incubate 5 hours to overnight at 37oC with mixing.  Four hours is sufficient for human IgG.
  8. For every 1ml of diluted antibody add 1.5ml 10mM tris-HCl solution, then centrifuge 1000g for 15 min at 4°C.
  9. Carefully draw supernatant off of papain pellet.

  • Smaller amounts of papain may be used, but longer digests are required and incomplete digestion may occur as a result.  For antibody quantities less than 10mg, use 500ul of slurry.

Fab Purification

Fab purification should be performed as per the instructions found in Fab purification using FPLC.