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Immunoprecipitation western blot troubleshooting



Immunoprecipitation western blot troubleshooting

In an immunoprecipitation-western blot I used rabbit polyclonal antibody and protein A agarose beads(protein A is covalently bind to beads) to pull down antigen complex. In following western blot I used rabbit polyclonal antibody as primary antibody and HRP-anti rabbit IgG as secondary. The result turns out to be many bands, including IgG bands. Is there a good way to do IP-W and get clean band?

I've never seen a perfectly clean IP-western, but I've done a fair few
that are pretty good. If you blot with an antibody from a different
species than the IPing antibody, and probe with a species-specific
antibody (not just an antibody raised to one species, which generally
cross-reacts; you need to use an antibody that has been actually stripped
of cross-reacting species), you can get clean results.

If you absolutely have no choice and must use two rabbit antibodies, you
might try covalently coupling the antibody to beads, instead of using
protein A; I've never tried this, but it might help, at least with the
heavy chain cross-reactivity.

Ian

I had the same problem. I was doing Western albeit without IP (rabbit
primary Ab, porcine anti-rabbit HRP-labelled secondary (DAKO Denmark),
ECL detection). The result was very similar. Seems the reason is not
IP but the antigen-primary Ab-secondary Ab interaction. The only
"clean" Western I have got was with biotinylated goat anti-rabbit
secondary antibody from Vector, streptavidin-AP coniugate and CSPD
chemiluminiscence (No affiliation to above mentioned companies).
Hope this helps,

Last update 02-Feb-2001, Rating of 1 votes.

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By biochemistvn on 27-Jun-2005
Hi,
I am performing the immunoprecipitaion but I am meeting a problem. Could you please give me some solutions?
My experiment was performed as following:
5 ug of the extract (containing antigen) + 1ug of primary antibody + 50 mM Tris, pH7.5, total volume of reaction was 300 ul, mixture was incubated at 4oC overning.Then add 50 ul of Protein G magnetic beads to the reaction mixture. This mixture was incubated continuously for 1 hour at 4oC. Mixture was applied onto the column (using MACS kit). Most of the interested protein was appeared in the Flow through. Nothing is in the wash, small amount of interested protein is in the eluent. That mean antigen could not bind to the primary antibody. Could you please give me some solutions to solve this problem?
Thank you very much!
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By From D.Kramer on 02-Feb-2001
If indeed these Abs are the only posibilities to use, covalent binding is a

good suggestion. I have had problems with the use of rabbit pAbs as well. I

have these suggestions



* Purify (one of) the antibodies using a CNBr column with the antigen.

Elute the antibody. * If this Ab is to be used to precipitate you can link

it to protA or protG beads (Pharmacia, NoAffiliationWith) eventually link

the Abs covalently prior to the IP. I do this with DMP (if you want the

protocol, ask me).

* Another strategy is to purify the pAb first, followed by coupling it to

CNBr beads (Pharmacia NAW).



Perform the IP



In case covalently linked pAbs are used, apply the non-reducing Laemly

buffer to the beads, boil it 2 minutes. If you want to have them reduced on

the gel, pipet the sample to a fresh tube, add 1% (f.c.) 2ME to the sample,

boil it for another minute and apply it to the gel. With DMP coupled beads

more Ab will be on the blot.



Stain the WB also with affinity purified pAb.



If you purify only one of the two Abs, use the purified bacht to stain the

blot.



D. Kramer
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