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biotinylated protein extracts and antigen-antibody stringency


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#1 Chelo

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Posted 13 September 2011 - 07:36 AM

Dear all,

I would like to set-up a test to detect a protein present in a whole protein extract and for which I have a polyclonal antibody. To this end, my idea is to biotinylate the whole protein extract and to fix the polyclonal antibody in an ELISA plate and to do an ELISA test, developing with streptavidin-HRP.
Now, I have two questions regarding this:

1) do you have any idea of how much extract should I test in a well? I can imagine that that would depend on the antibody afinity, on how rich the whole extract is in my protein and on the biotinylation efficiency. But since I am new in this world, I am just looking for a broad answer to get a kick-start point. Should I try 1 ug, 1 mg...a kg? :-)

2) because there must be protein complexes and aggregates that could make this test dirty, I would like to add some salt to the washing buffer to enhance stringency. So my next question is how much salt can an antibody-antigen interaction stand? I don´t want to make it too stringent!

Thank you very much in anticipation.

#2 PAO_ahac

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Posted 14 September 2011 - 02:48 AM

Coat the plate with your antibody and block it. You can test the blocking efficiency by reacting conjugate and washing. This will confirm your blocking step is ok.

For the samples run a dose response curve serial 1:10 or 1:3 dilutions cover several logs of concentration. Perhaps start at 10 mg/ml which should give you a very high signal.

Washing I would stay with just PBS with Tween. Upping the tween concentration will reduce background...but also some binding as well.

You will have to determine the correct conjugate concentration to use and, of course, optimize incubation times and temperatures. For washing you can include a dH20 rinse prior to your next reagent addition. Additionally, your wash solution can remain in the wells for 1 min prior to removal.

Hope these tips help.

#3 Chelo

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Posted 14 September 2011 - 03:11 AM

Thanks PAO_ahac!
My extracts are not that concentrated but I think I can start from 1 mg/ml.

#4 PAO_ahac

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Posted 14 September 2011 - 11:06 AM

Do a OD 240 to establish some starting point for concentration of the protein mixture and then go from that point. If you can purchase the purified protein that would be ideal to have some type of reference point.

If your MW is similar to albumin your elisa should be sandwich type for greatest sensitivity.




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