Hi Everyone,
Our group recently is trying to bind ProteinA on the acid-functionalized electrode through the EDC/NHS chemistry. We have seen the change through the activation of EDC/NHS but the following ProteinA-binding step just brought the impedance back to the same level of the unactivated surface (By EDC/NHS). Similarly, the other set immersed into PBS buffer presented the same impedance change which we thought may be caused by the hydrolysis. However, the success of activation has been proved by attaching other redox-active molecules containing primary amine. Does anyone have any ideas about this phenomenon? We presumed that the PI value of ProteinA was the issue but no matter we tried the high PI protein, avidin, or lower the PH, like 4, the supposedly increased impedance was not observed throughout the experiment.
Experimental details:
We activated the acid in the MES buffer at PH=6 and implemented the binding reaction in PBS buffer at PH=7.4. Binding and activation time is constantly 2 hours but longer time was also tested and no difference was realized
Will appreciate it if anyone could help with this problem.
Thanks.
Katja Werner Thanks for your anwser! I will go to double-check the details provided by the manufacturer and run a desalting column to see if it works.
There are many reasons, why NHS conjugations can fail. Katja Werner mentioned already the buffer of the protein, which should not contain any competing molecules. Some buffers and solvents (e.g., DMF) contain traces of amines, which deactivate your NHS esters. Phosphate, borate, and carbonate buffers should be safe. The pH of the protein solution should be in the range of 7.5-8.5. Conjugations are slow in neutral buffers. Often EDC has hydrolyzed in the bottle caused by air moisture and hence, became inactive. If possible, open a new bottle just before you start the experiment. In addition, the concentration of the protein (in this case protein A) should be 1 mg/mL or higher to be efficient. Unfortunately, this list is not exhaustive :-)
Katja Werner Hi Katja, I have checked the place where we bought the ProteinA. It says it is lyophilized but salt-free. So, maybe this is not the main issue. Thanks again for the answer.
Michael G. Weller Thanks for your time in answering this question. We used PBS buffer throughout all ProteinA binding trials. According to the vendor, there should be no primary amine in the formula. The PH for binding is 7.4 but we also tried 8, and 9. Still, no sign of binding was seen. In terms of EDC, we stored it in the freezer in the glove box and only took the amount we need for the experiment out right before the EDC/NHS treatment. We indeed used only 0.1 mg/ml for the conjugation trials and 1mg/ml should be a good try, but this could be the highest we could get according to the solubility of ProteinA (1 mg/ml). Thanks again for your valuable thoughts regarding my question.
Under these circumstances, I would buy a new vial of EDC. There is a pre-aliquoted "no-weigh" product available.
Michael G. Weller Thanks for the suggestion and we will try if it works.