Antibody Purification Methods, Techniques & Protocols



Antibody Purification on the Protein G Column (PDF file) - Protein G rather than Protein A is the column of choice for purifying mouse IgGs from ascites, because mouse IgG1 binds much (much!) better to Protein G, and most mouse mAbs turn out to be IgG1. However, Protein G cannot be used to purify from tissue culture supernatants containing fetal calf serum, because it has a very high affinity for the bovine IgGs in the serum. (Rat IgGs don't bind well to either Protein G or Protein A, and should probably be purified on an FcRn column.)

Antibody Purification - Frequently asked questions about antibody purification.

Antibody Purification by Affinity Chromatography - This protocol contains the following steps: Preparing the gel, Coupling the ligand, Binding, Elution.

Purification Of Anti Peptide Antibody By Claire Walczak January 1996

Coupling Peptides To Resin For Affinity Purification Claire Walczak January 1996 - We use Affigel-10, converting its functional group first to amino and then to iodoacetyl. You can also buy amino resin (it's more expensive) and start at step 6. All washes are performed on a glass-fritted filter funnel sucking until a wet cake is formed. Do not dry the resin completely or you will introduce lots of air bubbles. All reactions up to peptide addition are performed in the funnel by covering the spout with parafilm. This minimizes loss. Assume the affigel is a 50% slurry.

Affinity Purification of Antibodies - Including methods about conjugation of carrier (BSA) to peptide, Affinity Column Prep, and Affinity Purification.

Affinity Purification of Antibodies By Peter Vize - Different antibodies are eluted off affinity columns under different conditions. Most will come off in the glycine elution protocol outlined here. You can test if they came off or not by applying the "eluted" antibody to a small piece of nitrocellulose that has a spot of your protein on it and developing it by normal protocols. Glycine is the best option, as not only does it work with most antibodies it also causes less damage to the column itself.

Antibody Purication (Upstate) - Antiserum or Ascites by Protein A/G Chromatography

Antibody Purification Protocol - Antibody purification is possible because the cepharose beads are conjugated with protein which has an affinity under certain conditions to immunoglobulins. Following, you'll find a basic protocol for antibody purification either by Protein A or Protein G.

Antibody Purification on Protein G Column - Protein G rather than Protein A is the column of choice for purifying mouse IgGs from ascites, because mouse IgG1 binds much (much!) better to Protein G, and most mouse mAbs turn out to be IgG1.

Chicken Immunoglobulin Purification - Protocol including solution, equipment and step by step procedure.

Preparation of Site-Specific Antibodies and Affinity Purifications - Synthesis of N-terminal peptides of histones H2A, H2B, H3 and H4

Antibody Purification - This protocol includes an ammonium sulfate cut, affigel blue chromatography and affinity chromatography.

Antibody Purification Protocol - Ammonium sulphate precipitation is used to purify a protein (in this case an immunoglobulin) from a big volume of  liquid phase.

Published Antibody Purification Methods, Techniques & Protocols


Yueh SC, Lai YA, Chen WL, Hsu HH, Mao SJ.

An improved method for haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity chromatography in the presence of sodium dodecyl sulfate.
J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Jan 15;845(2):210-7. Epub 2006 Sep 7.
PMID: 16962396 [PubMed - indexed for MEDLINE]


Ishihara T, Yamamotob S.

Optimization of monoclonal antibody purification by ion-exchange chromatography. Application of simple methods with linear gradient elution experimental data.
J Chromatogr A. 2005 Mar 25;1069(1):99-106.
PMID: 15844487 [PubMed - indexed for MEDLINE]


Hylarides MD, Mallett RW, Meyer DL.

A robust method for the preparation and purification of antibody/streptavidin conjugates.
Bioconjug Chem. 2001 May-Jun;12(3):421-7.
PMID: 11353541 [PubMed - indexed for MEDLINE]


Cook CL, Pao W, Firca JR, Anderson BE, Fryer JP.

Simple purification methods for an alphagalactose-specific antibody from chicken eggs.
J Biosci Bioeng. 2001;91(3):305-10.
PMID: 16232994 [PubMed]


Dansithong W, Paul S, Kojima Y, Kamiya K, Shinozawa T.

A simple method for midkine purification by affinity chromatography with a heavy chain variable domain (VH) fragment of antibody.
J Biochem Biophys Methods. 2001 Mar 28;48(1):77-84.
PMID: 11282403 [PubMed - indexed for MEDLINE]


Choi J, Kim C, Choi MJ.

Influence of the antibody purification method on immunoassay performance: hapten-antibody binding in accordance with the structure of the affinity column ligand.
Anal Biochem. 1999 Oct 1;274(1):118-24.
PMID: 10527504 [PubMed - indexed for MEDLINE]


Vijayalakshmi MA.

Antibody purification methods.
Appl Biochem Biotechnol. 1998 Oct;75(1):93-102. Review.
PMID: 10214699 [PubMed - indexed for MEDLINE]


Yamaguchi T, Fujimiya Y, Suzuki Y, Katakura R, Ebina T.

A simple method for the propagation and purification of gamma delta T cells from the peripheral blood of glioblastoma patients using solid-phase anti-CD3 antibody and soluble IL-2.
J Immunol Methods. 1997 Jun 23;205(1):19-28.
PMID: 9236911 [PubMed - indexed for MEDLINE]


Hosomi O, Takeya A, Yasuda T, Yazawa S, Nakajima T, Kogure T, Iwai H, Kishi K.

Simple purification method of the antiphospholipid antibody from normal human plasma.
Exp Clin Immunogenet. 1997;14(4):281-5.
PMID: 9523164 [PubMed - indexed for MEDLINE]


Miyasato M, Tsuda S, Kitamura N, Shirouzu K, Nakama T, Sasai Y.

Purification of human blood eosinophils by a combination method using anti-CD16 monoclonal antibody, immunobeads, and Nycodenz density gradient.
J Dermatol Sci. 1995 Sep;10(2):118-29.
PMID: 8534610 [PubMed - indexed for MEDLINE]


Fernandes I, Cormont F, Latinne D, Bazin H, Takehara HA, Mota I.

A rapid and efficient purification method for horse IgG(T) using a rat monoclonal antibody.
Braz J Med Biol Res. 1994 Nov;27(11):2599-606.
PMID: 7549982 [PubMed - indexed for MEDLINE]


Gomes MA, Lima Wdos S, Pesquero JL.

A new method for bovine pepsinogen purification. Preparation of a specific antibody.
J Immunoassay. 1994 May;15(2):157-70.
PMID: 8040350 [PubMed - indexed for MEDLINE]


Rosenzweig HS, Ranadive GN, Seskey T, Epperly MW, Bloomer WD.

A novel method for the non-chromatographic purification of technetium-99m-labeled monoclonal antibodies: a study with B72.3 monoclonal antibody.
Nucl Med Biol. 1994 Feb;21(2):171-8.
PMID: 9234280 [PubMed - indexed for MEDLINE]


Naka A, Honda T, Miwatani T.

A simple purification method of Vibrio cholerae non-O1 hemagglutinin/protease by immunoaffinity column chromatography using a monoclonal antibody.
Microbiol Immunol. 1992;36(4):419-23.
PMID: 1406368 [PubMed - indexed for MEDLINE]


Skerra A, Pfitzinger I, Pluckthun A.

The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique.
Biotechnology (N Y). 1991 Mar;9(3):273-8.
PMID: 1367302 [PubMed - indexed for MEDLINE]


Zola H, Neoh SH.

Monoclonal antibody purification: choice of method and assessment of purity and yield.
Biotechniques. 1989 Sep;7(8):802, 804-8. Review.
PMID: 2561062 [PubMed - indexed for MEDLINE]


Mita S, Hosoya Y, Kubota I, Nishihara T, Honjo T, Takahashi T, Takatsu K.

Rapid methods for purification of human recombinant interleukin-5 (IL-5) using the anti-murine IL-5 antibody-coupled immunoaffinity column.
J Immunol Methods. 1989 Dec 20;125(1-2):233-41.
PMID: 2607155 [PubMed - indexed for MEDLINE]


Ford CH, Macdonald F, Griffin JA, Life P, Bartlett SE.

Immunoadsorbent purification of carcinoembryonic antigen using a monoclonal antibody: a direct comparison with a conventional method.
Tumour Biol. 1987;8(5):241-50.
PMID: 3329406 [PubMed - indexed for MEDLINE]


Alsenz J, Loos M.

Simplified methods for the purification, quantitation, and functional estimation of human complement C-1-inhibitor (C-1-INH) with a monoclonal anti-C-1-INH antibody.
J Immunol Methods. 1987 Jan 26;96(1):107-14.
PMID: 3805729 [PubMed - indexed for MEDLINE]


Phillips AP, Martin KL, Horton WH.

The choice of methods for immunoglobulin IgG purification: yield and purity of antibody activity.
J Immunol Methods. 1984 Nov 30;74(2):385-93.
PMID: 6438241 [PubMed - indexed for MEDLINE]


Hirabayashi Y, Suzuki T, Suzuki Y, Taki T, Matsumoto M, Higashi H, Kato S.

A new method for purification of anti-glycosphingolipid antibody. Avian anti-hematoside (NeuGc) antibody.
J Biochem (Tokyo). 1983 Jul;94(1):327-30.
PMID: 6619119 [PubMed - indexed for MEDLINE]


Taki T, Abe M, Matsumoto M.

Simple purification method for anti-glycolipid antibody using polystyrene latex beads coated with gangliotetraosylceramide.
J Biochem (Tokyo). 1982 May;91(5):1813-6.
PMID: 7096318 [PubMed - indexed for MEDLINE]


Page M, Audette M, Caron M.

A rapid method for the purification of antibody--enzyme conjugates.
Can J Biochem. 1979 Mar;57(3):286-8.
PMID: 108007 [PubMed - indexed for MEDLINE]


Folkersen J, Teisner B, Svendsen P, Svehag SE.

An immunoprecipitation-dissociation technique for large scale antibody purification and an antigen consumption electroimmunoassay for antibody quantitation. A model study with antibodies to pregnancy zone protein.
J Immunol Methods. 1978;23(1-2):127-35.
PMID: 29929 [PubMed - indexed for MEDLINE]





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