My his-tagged ELISA standard protein was received in at 5 mg/mL and I have made aliquots of it to reduce freeze thaw cycles. To create the highest standard, I must make a 1:400,000 dilution and from there I make 1:2 serial dilutions for the rest of the standard. OD readings for the high standard range from 1.5 and 2.5 each run and are sometimes within the linear range of the curve and sometimes above the linear range showing poor repeatability. What is the best storage concentration and storage solution to reduce the pipetting error from the large initial dilution and reduce and destabilization and aggregation during freezing and thawing?
Carrier proteins can be essential for your positive standards in ELISA, with casein (Na caseinate really) or BSA being the most common. In addition, surfactants that aid in ELISA plate blocking ( Tween 20, Triton X-100, and CHAPS) can also help with standard stability. That said I used to work with a "limiting antigen" HIV ELISA which had no need for carrier proteins even though it required std dilution to near a zero signal! Good luck!
It's difficult to answer that question without knowing what protein it is and in which buffer it is stored. Concentration alone is not the only parameter to consider. You need to test it in a concentration range which allows good stability after freezing/thawing (with or without protecting proteins like BSA) and convenient dilution factors, ideally in and different buffers with additives. In general most proteins are not "happy" below 50-100µg/ml without e.g. BSA, because of adsorption to the vial, you can observe significant reduction in its concentration, not necessarily because of stability issues.
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