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Coating Buffer for Sandwich ELISA


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#1 rensky

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Posted 27 January 2005 - 12:37 PM

Hi guys!

I've been trying to coat a custom peptide onto Immulon plates because I am trying to develop an assay. Sometimes I get signal, sometimes I don't.

I'm wondering whether it will be because the coating of the "sample" is not done well?

I use 1x PBS + 0.02% Tween-20 for coating.

Any other coating buffers out there? How would I know which type of buffers are best for my peptide, sample, antibody, etc?

Thanks!

#2 jadefalcon

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Posted 28 January 2005 - 03:51 AM

Hi!

I used a carbonate-coatingbuffer:

0.1 M Na2CO3
0.1 M NaHCO3
pH=9.5

coated 100l/ well of a 96 well plate (medium binding capacity, but maxisorp plates worked equally well) at 4C over night.

worked for all kinds of proteins. if it doesn't work with your protein, may changing the pH wpoulod be a good idea.

mike
--- He who finds typos may keep them! ---

#3 rensky

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Posted 04 February 2005 - 04:05 PM

I was told that I shouldn't add blocking buffer when coating capturing antibody because the proteins in the blocking buffer will bind to the Immulon (coated) plates?

Does this mean that I should only block once in the protocol and just use phosphate buffer to dilute any antibodies I need to use in the protocol?

Thanks!




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