I´m trying to coat an ELISA plate with a protein which pI is ~5.20. I´ve been using a bicarbonate/carbonate buffer for the coating process, however it seems that my protein does not attach to the wells.
Should I choose another buffer, if yes, which one?
Hi. Proteins bind to plastic primarily through hydrophobic interactions, so the pH or buffer is probably not to blame. Things that will interfere with the binding are detergents and other proteins/peptides. Things that increase the binding would be high salt and high T.
You can always check the coating efficiency by doing a protein assay on your coated wells.
In my experience, the type of plate you are trying to coat your protein to makes a big difference. I've had the best results with NUNC maxisorp plates. I've tried using uncoated plates, and the only way I was able to attach my protein was by using methanol fixation.
Avoid the maxisorp plates. Most of the binding of a protein to the plastic surface is done by hydrophobic interactions. This will work in basic, neutral an acetic buffers as well.
Protein concentration and fixation times also affects on the attachment of your protein to the wells. What protein concentrations and fixation times have you been using?
Usually 1- 10 µg/mL is sufficient. It needs to get always optimized by checkerboard titration
If the carbonate Buffer (pH9.5, BD recipe in most elisa sets) doesn't work, you can try standard PBS (pH 7.4) and phosphate coating Buffer pH6.5 (recipe is in the manual of BD Opteia murine IL-12 p40 ELISA Set). Usually one of them works.
If not, you might want to make a sandwich ELISA and look for another antibody to your protein for coating.
I usually express recombinant proteins with a tandem Flag-His tag, and after the (cheap) Ni-NTA purification (not per se necessary to capture your protein with flag, but you can store it much better than lysates), you can capture them on Anti-Flag-M2 coated plates (coat them with 0,5µg/ml Anti-Flag-M2 in pH9.5 coating buffer)
Use 0,1M NaHCO3 (overnight [no more that 16h] at 4c) also, importantly, use azide and proteinase inhibitor.
pH of coating buffer will be 8.2 (don´t need to adjust, but prepare it fresh every time to avoid pH shifts)
My original question was about bicarbonate/carbonate buffer pH9.6 (http://www.abcam.com/ps/pdf/protocols/Direct%20ELISA%20protocol.pdf). The one that i´m using now (0,1M NaHCo3), has a lower pH (8.2), and it seems to work with acidic proteins.
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