Lab Manual
biological reagents
Cell Free Systems
In Vivo Systems
misc methods

> Summary of Rabbit Immunization Procedures

    > Affinity Purifying Antibody from Rabbit Serum
    > Purifying Antibodies Made using a GST-fusion Protein


Purifying Antibodies Made using a GST-fusion Protein

I. Before you begin:  

  • Grow up cells expressing GST alone and cells expressing your GST-fusion 
  • Lyse in normal manner (e.g. French Press) 

II. Cross-linking GST and GST-fusion to Glutathione-Sepharose:

  • Equilibrate 1-ml of Glutathione-Sepharose 3X with 10mls of whichever buffer was used for lysis 
  • Add up to 100 ml of lysate containing either GST or GST-fusion -incubate 1 hr. @ 4ºC 
  • Wash again 3X with 10 mls of:

    bullet20 mM HEPES-NaOH, pH 7.5

    bullet150 mM NaCl

    bullet1 mM EDTA

    bullet0.5% Triton X-100 

  • Wash resin 2X with 10 mls of: 

    bullet200 mM Borate-NaOH, pH 8

  • Add 2 ml of FRESH DMP solution:

    bullet200 mM Triethanolamine-Cl, pH 8.3

    bullet8 mg/mL dimethylpimelimidate-Cl

    bulletmix continuously for 30 mins @ room temp

  • Terminate cross-linking with the addition of 10 ml of 200 mM ethanolamine-Cl, pH 8.2 
  •  Wash twice with 2 ml of 100 mM glycine-Cl, pH 2.5 
  •  Wash twice with 10 ml of TBS (20 mM Tris-Cl, pH 7.0; 150 mM NaCl) 
  • Store in 0.02% azide until you are ready for step III 

III. Purification of Antibodies:  

  • Wash columns with 20 ml TBS 
  • Combine 2 ml of serum with 2 ml TBS and incubate with GST-linked column for 1 hr. at 4ºC 
  • Take eluate from GST-linked column and combine with a 5 ml wash with TBS then incubate with GST-fusion linked column -incubate 1 hr. at 4ºC 
  • Wash column with 20 mls TBS 
  • Wash column with 10 mls 0.1X TBS 
  • Elute with 5 ml of 0.1M glycine-Cl, pH 2.5 (take 1 ml fractions) 
  • Adjust pH of elution fractions to pH 7.0 with Tris-Cl, pH 8.0 (~100 ul)
  • Regenerate columns with 0.1M phosphate solution, pH 12