Hi, I am purifying a synthesized GST tagged membrane protein with the following condition:
Crude preparation:
100ml culture was centrifuged at 12000xg for 15 min
The pellet was resuspended with PBS buffer with 0.1% Tx-100.
The lysate was sonicated at 30% A for 8.5min, 15s intermittently
The lysate was centrifuged at 12000xg for 15 min
Supernatant was filtered and protease inhibitor cocktail was added
GST purification
10ml of sample was injected to 1ml Cytiva GSTrap column
The resin was washed with 20CV PBS buffer
Protein was eluted out with 50mM Tris HCL with 10mM GSH at a gradient
My question is:
Why are there multiple other bands apart from my target protein (80kDa)? Do I oversonicate causing the GST fusion protein to rupture? Or is there are any other reason?
Some of the extra bands could be proteolytic products of the desired protein. This problem could be reduced by including a protease inhibitor cocktail in the lysis buffer, rather than later.
Some of the bands may be premature termination products containing the GST tag (assuming it is N-terminal).
If the Triton X-100 detergent you are using is old, it probably contains peroxides that are degrading your protein. Get some fresh detergent, preferably Surfact-Amps-100 from Thermo-Fisher Scientific.
Since you are working on a membrane protein, you need to keep a detergent in the buffer at all times to keep the protein in solution. You may be losing most of it when you do the column washing and elution steps without detergent, so you are mostly getting the minor contaminants off the column.
Hi,
The 25 Kda band looks like GST alone. Could it be that you have a subpopulation of transformants that don't contain your target protein sequence? If so, streak a plate and pick a new colony.
If this is a heterologous protein, did you optimize the codons for your host? There are rare codons in E. coli that can inhibit translation because there is a low population of the tRNA for that codon.
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