ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera.
Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33 kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time.
Washington University School of Medicine, Department of Pathology and Immunology, Division of Laboratory and Genomic Medicine, 660 South Euclid Avenue, Box 8118, Saint Louis, MO 63110, USA. crimmins@pathology.wustl.edu
Dan L
DL
Crimmins
Nancy A
NA
Brada
Christina M
CM
Lockwood
Terry A
TA
Griest
Rachel J
RJ
Waldemer
Mark A
MA
Cervinski
Matthew F
MF
Ohlendorf
Jay J
JJ
McQuillan
Jack H
JH
Ladenson
D000818:Animals; D000906:Antibodies / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003001:Cloning, Molecular; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000097:blood* / Q000276:immunology; D007106:Immune Sera / Q000737:chemistry*; D007118:Immunoassay; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000276:immunology; D012756:Sheep
D000906:Antibodies; D000939:Epitopes; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase