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Sample Antigen in Extraction Buffer vs. Purified Antigen Standard in PBS


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#1 WolfeMD

WolfeMD

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Posted 13 December 2009 - 02:57 PM

I've designed a Sandwich-type ELISA for a plant HSP. I had some trouble getting it working at first, but things have really been going well... so far. I've so far done tests of optimal dilutions of the reagents by comparing different dilutions against standard dilution series of purified target protein in PBS. The full range that I have tried works for most dilutions from 0.125 ng/uL to 8 ng/uL.

My question for the forum is this:

What I ultimately want to do is extract total cellular protein from tissue samples and test those extracts for the target protein. Should I anticipate any trouble with this? Here is my extraction buffer:

2X Sample Buffer (25ml)
i. 6mL 1M Tris (pH=8)
ii. 1.0 g DTT
iii. 1.6% Triton X-100 (0.8% in 1X working dilution)
iv. 15g Sucrose
v. 0.06g E-Aminocaproic Acid
vi. 0.018g Benzamidine

I normally get about 2 ug/uL of total protein (as per a modified Bradford assay I use). In western blot I have seen no problems with non-specificity and my two antibodies used in the ELISA come from Rabbit and Hen and recognize different epitopes... so they should be perfect for ELISA.

See a more in depth description of my protocol here: http://www.protocol-...showtopic=11709

I'm looking for any advice that can help me anticipate trouble.


I have two other questions:

What does the signal-to-noise ratio (sample OD / blank OD) tell me that blanking does not?
How high of a signal-to-noise ratio should I be expecting? I got a range from about 1.25 to about 11.0 across the target protein dilution series.

Cheers,

M




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