I want to perform few studies on therapeutic protein available in market. But as control, I have to use only buffer without protein in it. Since I am not sure of exact composition of buffer, I can i remove only protein from that formulation, so as to get buffer without protein. Addition of some chemical is not good choice as that will make buffer composition different from buffer in which protein is present. Any suggestions welcome...
Use ultrafiltration with a membrane having molecular weight cutoff smaller than the protein. The filtrate will contain the low-molecular weight components of the solution, leaving the protein behind.
Use ultrafiltration with a membrane having molecular weight cutoff smaller than the protein. The filtrate will contain the low-molecular weight components of the solution, leaving the protein behind.
I concur with Adam's method as this is what we use in the laboratory for "reverse" analysis. You need to ensure that you have all appropriate controls in place, such as water in the ultafiltration device, PBS in the ultrafiltration device, etc. You must prove that your device has not added anything to the buffer you isolate. Furthermore, there are many materials that do not come through ultrafiltration membranes and some items will be altered if you have the Donnan (Gibbs-Donnan) effect taking place.
thanks Adam and Steven for valuable suggestion. Yes this can be tried. Only problem is loss of some salt/excipient to filter and that may results in difference in buffer composition wrt protein formulation.
The ultrafitration method is probably your best bet, despite the potential loss of salts. To minimize the salt loss, you could try prewetting the filter with a reagent you suspect is in the buffer - but that could also slightly dilute the buffer. Chances are the modifications to the buffer from ultrafiltration will be minimal and negligible and good enough to use as a control for comparison to the active therapeutic (this is assuming there are no complications such as Steven has mentioned).
Have you tried contacting the manufacturer of the therapeutic? Sometimes they will hint at what the proprietary buffer is, or suggest a solution to your issue. Could take a lot of phone calls until you reach the right technical assistant, but it is worth a shot.
To test whether salts have been lost during ultrafiltration you could test for a decrease in conductivity and osmolarity, or have the samples analyzed for the presence of specific ions by atomic absorption spectroscopy or ICP-MS.
Thats good idea... i can test for change in conductivity.... Thanks...
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