Hello,
I am trying to purify a GST tagged protein of 16kDa (Total MW with GST is 43KDa). I used affinity chromatography with GST beads to initially purify my protein and it yielded a fairly pure protein with decent concentration.
My problem starts when I am using size exclusion chromatography to further purify my protein. I used 50mM Tris pH8, 150mM salt, and 1mM DTT as my running buffer but to my surprise I see no peak at the chromatogram. Just a flat straight line. I tried twice and changed the composition of my buffer to 50mM Tris pH8.5, 300mM salt, and 1mM DTT but still the same problem persists. I see no protein at all.
The pI of my protein with GST is 5.87.
What could be the reason for this loss of protein? Is my protein aggregated and having some non-specific interactions with the column? Is my protein getting precipitated inside the column?
Can I just eliminate the gel filtration step and carry on with some other purification step like IEX?
What gel/matrix are you using? Are you measuring the protein through a flow cell in which case have you checked the cell/uv bulb is capable of measuring the column output?. A lot of gels separate very differently depending on the shape rather than the size of the protein how are you checking the protein levels in the collection tubes?
I am thinking about 2 things:
- Have you checked the maximum absorption wavelength of your protein. OD280 nm commonly is used for detect protein but in many cases, 230 nm shows the better result.
- Be careful with protein concentration. Gel fitration can dilute your protein concentration 10 times. Thus, if your protein concentration is not high enough, the monitor can not detect the protein after gel fitration. In this case, you must concentrate the protein first
Could the protein be insoluble? then it would be filtered out at the top of the column. Hint: always spin your sample before loading it onto a column. Also, make sure your protein is soluble in the running buffer.
Whether you can use IEC on your protein without GC depends mostly on the buffer you have that protein in. High salt (e.g., after AS precipitation) prevents binding. In that case, hydrophobic interaction would be a better choice.
Hello everyone,
Thank you very much for your help and suggestions. The machine I was using had some issues. I tried my sample in a different ACTA machine and it works fine with the current buffer. The protein is perfectly soluble and is highly pure based on both the chromatogram peak and SDS PAGE gel. In last two days, I have searched every possible scientific reasons for my protein being lost in the system but it did not cross my mind that there might be a problem with the machine. I apologize for my naivety.
Again, I want to thank everyone who tried to help and solve this problem. :) :)
Hello, Sayan. I hope you've resolved your problem. I have had the same problem. If you have some suggestion, I would be very thankful to know, because it could help me too. ( Sayan Chakraborty )
Hello Glaecia,
The problem was with the akta system we were using. I tried it in a differ akta purifier and it worked fine. sorry, I can’t help you much. But check if your protein has any nonspecific interaction with the column or it is crashing into the column.
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