My dear all,
I purified trx tagged protein, but to cleave the tag I has to use enterokinase which works in the Tris buffer. My protein was in phosphate buffer.how can I transfer my desired protein to Tris buffer? Some one please help.?
You can use desalting columns or dialysis bag to exchange the buffer ions. Please check.
You could use dialysis tubings or dialysis cups (if you have a small amount of sample). In our lab, we dialyse ~1 ml of protein sample in 1000 ml of buffer (with gentle stirring). In your case, you need to dialyse your sample in a Tris buffer. Then, change to a fresh 1000 ml buffer after one hour of dialysis. Just ensure that you are using a tubing with the correct MWCO (to ensure that your protein is retained in the tubing).
You could also try using buffer exchange columns. In this case (depending on your product info sheet), you will dispense your sample into the column and centrifuge the column at a certain speed for a certain amount of time. Then you will dispense your Tris buffer into the column and repeat this step for a couple of times. I don't really use this method- so you will have to check with others/online.
Size exclusion methods (e.g. desalting column or FPLC) or dialysis methods are the most common. You can also do repeated ultrafiltration (centrifugation with a membrane of a desired MW cutoff value) if you are desperate, though it is far less time efficient, it will work.
Out of curiosity, was there a reason you can't use Tris in the earlier steps? I am not familiar with trx tags. You can also check with the manufacturers of both the enterokinase and the trx purification method and see if there is another buffer that will be suitable to both methods. It's rare for a process to only have one possible buffer that works.
You can buy desalting spin columns from thermosvientific. You just add your sample to the top of the column after equilibrating it, and spin. The protein comes out in the flow through.
You can also get free concentrator samples from EMD Millipore with different MW cutoff. You just dilute your protein into the desired buffer, concentrate, dilute again and repeat
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