alternative coating buffers for ELISA - anything else i can use aside from PBS and carbonate/bicarbonate? (Jun/18/2008 )

for certain ELISA experiments we do, we need coating buffers that have minimal salt content (esp. salts which yield small positive ions like Na+ and K+ in solution). i'd like to try Tris-EDTA pH 7.4, Tris-EDTA pH 8.0 and distilled and/or deionized H2O. has anyone tried these as coating buffers and succeeded?

2nd question: can i use dH2O instead of PBS for the washing steps in ELISA?

3rd question: when doing sandwich ELISA, which fragment of the capture antibody binds to the PVC plate? is it the Fc region (as indicated in most schematic diagrams)?

thanks very much everyone!

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See if you find an alternative buffer here:
http://search.vadlo.com/b/q?sn=158621799&a...ELISA&rel=0
..

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also please watch the pH. carbonate buffer's ~9.4 usually.

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On occasion we have had to use ethanol or methanol.

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cellcounter: thanks! i did look at that link, but all i've seen so far are PBS and carb/bicarb. there are also some commercially available ELISA coating buffers mentioned but i do not know what they contain exactly, or if it's even possible to find out.

aimikins: thanks for the reminder. we'd like to keep the pH at 7 to 8.

swanny: interesting... do you mind sharing more? what concentration of ethanol or methanol did you use, and would this be applicable to most proteins?

thanks again, everyone.

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You can use carbonate, tris, phosphate as long as pH is greater than the pI of the protein. Usually 8 or so for abs. You can also shock ab with low pH glycine for 5 min prior to dilution in your coating buffer to open up the protein...and get more stuck on the plate. If storing plates block with bsa, decant, then coat with sucrose for 30 min, decant and allow to dry inverted. Some people do not use BSA and use milk proteins or proprietary 'blockers' from Pierce, KPL, Surmodics.

You can wash with dH20; I have only seen few protocols with this. Most protocols are some buffer with BSA +/- surfactant (ie tween 20)

Ab arrangement on the plate is random.... depending upon conditions most will be to Fc.

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Alcohol coating may be beneficial for some lipids (I used it for cardiolipin) or proline-rich peptides (gliadin peptides for coeliac disease). The use of alcohol is the exception that proves the main rules of coating, however, so it most probably won't work for most proteins. If you want to give it a try, you might have to coat with a range of percentages, from 100% down to, say, 50% (complete guess there, but you might just have that one stoopid protein that likes it!!!!).
Try it, but don't get your hopes up too high.

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Hi frogattack,
I have tried coating my protein in bicarbonate buffer (pH 9.5), PBS and Tris (pH 7.4) and i couldn't see any difference
i think you can use any buffer for coating but not sure about pH so you can try at pH 7.4
also few protocols say to use dwater for washing so i think you can use it
all the best
Leelaram

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