affinity purifcn. Ab
ED at micro.uct.ac.za
Fri Jun 25 03:40:51 EST 1993
> We have done two batches of affinity purification of an antiserum on an
> immobilized fusion protein (bacterial expression and transfer after SDS PAGE)
> and the resulting antiserum works in Western blots.
> We will do some fluorescence studies now and see what that gives.
Glad to hear it...!
Can't find detailed protocols - but are extremely easy.
Lunchbox technique for pre-absorbing AS prior to Westerns, etc: Soak a
10x10 cm piece nitrocellulose in extract of tissue, plant, bug, bacteria,
whatever, NOT containing protein of interest, in a lunchbox / freezer
box (hence name of technique) 20 min or so. Wash lightly,
block thoroughly, add AS DILUTION: ie, same mix you would add to your
blot. Incubate 1hr or so, simply pour off AS soln onto blot. All Ab to
contaminants/ host proteins should have been removed.
This has worked very sucessfully with detection of plant virus coat
protein bands against a background of whole plant, using AS that reacted
with EVERYTHING before absorption; also with AS raised against
purified proteins either from, or cloned into, E coli: in
latter case, as anyone who has done it knows, when you do a Western, ALL
te bands light up, as rabbits are immune to E coli and related gut
microflora, and no-one thought to tell you...! In this case, a sonicated
soup or French-pressed mush works fine. Another, simpler, trick is to
dilute the detecting Ab into a suspension containing the same things you
would adsorb onto NC - but then re-use of the Ab is difficult, as
proteases in the suspension eventually eat the Ab.
Can use the same technic to mass-absorb / elute Ab to a particular
protein, without having to go to the trouble of making up a particular
immunoabsorbent: soak NC or other memb in protein of interest, wash,
block, soak in AS. Wash thoroughly, then elute Ab with preferred elution
mix (I use 0.1M Glycine/HCl/0.15M NaCl pH 2.9). Can repeat the
absorption/elution several times, and yield is quite high - certainly
enough for labelling specific Ab for immunofluorescence, ELISA, etc.
Have used it in our labs to make monospecific Ab to plant viruses, and to
E coli proteins or proteins cloned in E coli, as long as one has a
background free of the protein of interest.
Can also combine two techniques: pre-absorb AS with memb with complex
mixture NOT containing prot of interest, then pour off AS onto memb with
complex mixture CONTAINING prot of interest, preferably at highish concn.
First absorption takes out Ab reacting with "host protein", in second,
what is left is hopefully relatively monospecific, and can be eluted as
above, for labelling, etc.
Very simple, very easy, VERY CHEAP!!! - and published in such an obscure
place you'll never find it, but here it is anyway:
EP Rybicki, MB von Wechmar and JT Burger (1990). Monospecific antibody
preparation for use in the detection of viruses. pp. 149-153 in "World
Perspectives on Barley Yellow Dwarf" (PA Burnett, ed.); CIMMYT, Mexico
Hope is useful.
| Ed Rybicki, PhD | "Lord, won't you buy me |
| (ed at micro.uct.ac.za) | |
| Dept Microbiology | A Mer-ce-des Benz..." |
| University of Cape Town | |
| Private Bag, Rondebosch | |
| 7700, South Africa | - Janis Joplin |
| fax: 27-21-650 4023 | |
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