I am measuring Hsp 27 protein by sandwich ELISA and I will use monoclonal anti-Hsp 27 antibody and I need to dilute it in a buffer. The protocol says to use bicarbonate as a coating buffer. However it has PH =9.6 . Shall I prepare bicarbonate in a PBS or in distal water?
You need to have as much as possible hydrophobic sites in your protein. The binding to the polystyrene surface of the micro-titer plates or latex particles (which are also made from polystyrene) is done via hydrophobic interactions. At a basic pH you have this situation in your protein. 0.1 mol/L carbonate buffer pH 9.6 get you into this conditions.
Simply mix 0.1 mol/L NaHCO3 with 0.1 mol/L Na2CO3 under permanent pH control until you get the pH 9.6.
Hi Raha, the NaHCO3 coating buffer should be prepared in distal water not in PBS. PBS is phosphate buffer solution which is already a buffer by itself. after preparing the NaHCO3, you have to adjust the PH of the buffer as it will be more into acidic range. Wish all luck doing your ELISA
After you prepare the buffer, store it in well-sealed aliquots at 4C.
Hi Raha, I can confirm what Hani said, bicarbonate buffer should be prepared in distal water not in PBS. Also, remind that, when using bicarbonate buffer for coating, background are slightly more elevated that when using PBS
You need to have as much as possible hydrophobic sites in your protein. The binding to the polystyrene surface of the micro-titer plates or latex particles (which are also made from polystyrene) is done via hydrophobic interactions. At a basic pH you have this situation in your protein. 0.1 mol/L carbonate buffer pH 9.6 get you into this conditions.
Simply mix 0.1 mol/L NaHCO3 with 0.1 mol/L Na2CO3 under permanent pH control until you get the pH 9.6.
Thanks all for your help, but I needed to have an extreme PH to stretch the antibody and make it bind to the plate properly. the buffer I prepared was 50mMol of bicarbonate.
If you are going to extreme pH, you are on risk for (here: basic) hydroylsis. If you need a changes in the conformation of the antibody, use higher salt concentrations: e.i. 0.3 ... 1 mol/L NaCl, 1 ... 5 mol/L Urea, 1 ... 2.5 mol/L guanidin -HCl .... (the last one will change the pH)
Agree with Stephan and follow sigma protocol...https://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html
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