Search

Western or Enzyme-Assisted Imunoelectro-Blotting (IEB)

Elution of Specific Antibodies from Blotted Proteins

Molecular Biology Techniques Manual

Third Edition

Edited by:

Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki

 

ELUTION OF SPECIFIC ANTIBODIES FROM BLOTTED PROTEINS

Copyright Ed Rybicki, 1997

Contents

INTRODUCTION

PROTOCOL

REFERENCES

 


INTRODUCTION

Western blots often suffer from annoying background problems, mostly due to antibodies reacting with proteins which contaminated the inoculum used to raise the antiserum: failure to absorb out antibodies to E coli proteins, for example, can lead to specific reactions being lost in a blot of total E coli proteins in which one is trying to detect a specific product.  This is due simply to the fact that experimental animals are naturally immune to E coli - and to a large variety of other microorganisms and food substances.

A very simple method for removing unwanted antibodies from a serum is outlined in another section; this protocol will describe two methods for the preparation of "monospecific" antibodies from a serum with a wide range of reactivities.

The first technique was described by Olmsted et al. (1981) and Rybicki (1986).  It consists of  the electrophoretic blotting of a mixture of proteins, the excision of a specific protein band from a blot, and its use as an immunoadsorbent to purify specific antibodies which could then be eluted and used in specific westerns or ELISA.  The advantage of the technique over other immunoabsorbent techniques is that it allows the purification of usable amounts of antibodies (eg: sub-microgram) from very small amounts of a specific protein in a complex mixture.

The second was described by Rybicki et al. (1990): it consists of a subtractive technique, whereby a mixture of  proteins containing the one of interest (preferably at high concentration) are blotted onto a membrane (without electrophoretic separation), and then used for immunoadsorptive purification of specific antibodies from a serum from which "contaminant" reactive antibodies have been removed by adsorption to a membrane covered in a protein mixture lacking the one of interest.  This kind of procedure could readily be used to purify specific Ab for a protein cloned in E coli, with an extract of the bacterium which did not contain the clone being used for the subtraction.  The advantage over the electrophoretic method is that larger amounts of both antigen are used, and larger amounts of antibody can be purified (eg: 1 milligram from one elution from blot).

PROTOCOL

Electrophoretic Method

  • Electrophorese protein mixture of interest in as many lanes as possible across an SDS-PAGE (normally do several gels).

     

  • Electroblot gel(s) onto nitrocellulose or other membrane.
  • Locate protein of interest by cutting a strip of membrane off each side of big blot(s), and staining with Ponceau S or other reagent (or even doing immune reaction and stain).  Align strips with larger blot, and excise region corresponding to the band on the big blot(s).  Block as for normal western blot.

     

  • Use excised horizontal strip(s) to capture specific antibodies in normal western blot antibody incubation step: perhaps use higher concentration of serum, and pool all strips together.  Wash normally, and rinse briefly in distilled water and drain well.

     

  • Elute antibodies from strip with (for example) 0.1M glycine-HCl, pH 2.9: soak strip in (eg:) 10 ml buffer for no more than 5 min, then remove to wash buffer (eg: PBS-Tween pH 7.4).  IMMEDIATELY neutralise the glycine-HCL eluate with predetermined amount of 0.5M NaOH.

     

  • REPEAT antibody attachment / elution step as many times as is feasible (up to 5 in my hands).

     

  • POOL antibody eluates, add blocking buffer, use undiluted in westerns or ELISA tests.

     

NOTE: One may purify Abs capable of reacting with a single protein band from sera reactive with total plant or E coli proteins by this method - one may also use the Ab to subsequently purify protein from a complex mixture by immobilising the Ab on a membrane or other surface.

 


"Lunchbox" Method

See also HERE

  • ADSORB proteins to a piece (eg: 8x10 cm) of nitrocellulose or other membrane (eg: polyvinylidene fluoride) by simply incubating the membrane (room temp, gentle shaking) in a protein extract (eg: 25 ml of plant sap containing a plant virus) in a Tupperware or other freezer or lunchbox
    (NB: DO NOT USE blocking buffer to resuspend protein!!!!)
  • WASH membrane 3x5 min in western blot washing buffer (eg: PBS-0.05% Tween 20 pH 7.4 OR saline-Tween), then block as for a western (eg: in PBS-Tween / 2% non-fat dry milk).
  • ABSORB the dilution of serum to be used (eg: 1/50, in blocking buffer) by incubating it with a similar blot NOT containing the protein of interest (eg: plant sap WITHOUT the virus); 1-2 hr at room temp, with shaking.
  • POUR absorbed serum directly onto washed, drained specific blot membrane; incubate as above.
  • WASH blot as above.
  • ELUTE Ab as for electrophoretic technique, BUT in larger  volume of eluant buffer (eg: 25 ml).
  • Re-use blot as required.

NOTE: as above; Ab purified can be readily used to purify Ag from crude mixtures by a reversal of the technique (eg: adsorb Ab to blot, fish out Ag, elute).

REFERENCES

Olmsted, JB (1981).  Affinity purification of antibodies from diazotised paper bots of heterogeneous protein samples.  J Biol Chem 256: 11955-11957

Rybicki, EP (1986).  Affinity purification of specific antibodies from plant virus capsid proteins immobilised on nitrocellulose.  J Phytopathol 116: 30-38.

Rybicki, EP, von Wechmar, MB and Burger, JT  (1990).  Monospecific antibody preparation for use in the detection of viruses.  pp. 149-154 in: "World Persepctives on Barley Yellow Dwarf" (Burnett, PA, ed.); CIMMYT, Mexico DF, Mexico.