Using H2O to wash ELISA plate? - (May/14/2008 )

Hi Guys,
I wonder why PBS is used. Is it for the protection of antibody and antigen conformations so that Ab/Ag binding can effective occur? Could anybody give some links re the influence of osmosis on protein functions in general (or more general, biomolecule function)?
Hope this seemingly stupid question can induce some valuable comments.
Thanks, Hanhan

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I would guess antigen-antibody binding requires some salt.

While this is no great reply, at least I would think it has nothing to do with osmosis. Let us wait for some valuable comments
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Thanks. Your comments are valuable to me! . I didn't put it clearly, I guess. I mean osmolality (I'm not even sure if this is accurate). Just like cells, which require a physiological osmolality, or ionic strength (i.e., isotonic environment) to survive, I guess many enzymes, antibodies, other proteins, and non-protein macromolecules may also require certain osmolality in order to function normally.

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Of course appropriate salt concentration (usually close to physiological conditions) is important for protein structure, stability and function. Proteins have exposed charged residues and the salt provides counter-ions, which decreases the electrostatic free energy of the protein.

Another important function of PBS is its buffer capacity - PBS maintains physiological pH, which is also very important for structure and stability, as protein charge is dependent on it.

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Theory is theory. Only experiment tells the truth. Your protein might not change a lot in a few minutes from pH7.4 in buffer to pH6 in water. It may have already bind tightly with the necessary ions for its conformation and be able to tolerate a few minutes of water wash. My friend works on heparin binding protein ELISA. He uses all water to wash the plate now after he did a comparison run. It may be protein dependent, but if you wash one plate with .05% Tween20 water and the other with .05% Tween20 PBS and get the same exact standard curve, why not use water and it would be way more convenient from now on, at least for your protein or antibody.

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True.

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Interesting! That lowers the cost!

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Hi,
protein do precipitate in water
but in solid phase where it is already bound to the plate water wash may not be able to precipitate it
and also as earlier suggested the protein might already be bound with the counter ions which might not be washed away within a wink of time
hope this adds up to the discussion
Leelaram

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hi,
every time u use different antigen or different antibody, may be u need to check with water washing step before u put it in standard protocols.

in research, we take several precautions to have sensible results. y to take unnecessary efforts in reinventing the wheel. changing protocols will take ur time away. may be in that time u can find something new or interesting or u can focus on solving other real problems.

ofcourse i agree with water wash as long as one have patient to repeat the experiment in worst case scenario.

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