Hello,
I am expressing an integral membrane protein (MW 51kDa) in Hi5 insect cells. I initially purified the protein by Nickel-His, concentrated it and subjected it to Size Exclusion Chromatography. I am getting a weird shaped gel filtration profile. The chromatogram is broad and asymmetric ( see attached picture) with no defined peak. My protein seems to be from fraction 14-22. I am also attaching the gel picture. SEC buffer composition: 25mM Hepes pH 7.8, 300mM NaCl, 1mM TCEP, 0.05% DDM-CHS. I see broad asymmetric peak when I use DDM instead of DDM-CHS. I am using superose 6 10/300 column with a flow rate of 0.3ml/min.
Is it possible that my protein is unhappy in the detergent(s) I am using and getting unfolded or it has oligomeric species but can not be identified due to low protein quantity? There is no prior knowledge about the protein oligomeric states. Any suggestions/ comments on what is going on?
Thank you!
If your protein was pure and existed as either a monomer or in a single oligomeric state, you might expect to have a symmetrical peak. From the gel, it is apparent that the protein is very far from being pure. The asymmetric peak therefore reflects the fact that there are many proteins of many different sizes eluting from the column.
The protein doesn't have enough purity as seen from the gel image. Besides, concentrating this after Ni-NTA, concentrates the impurities (undesired proteins) as well. As a result, SEC produces asymmetrical broadened peak. You might try SEC before concentrating the protein, and collect the elution fractions. Run the fractions (if not known) in an SDS-PAGE to know which fraction contains your desired protein. Once that's known, collect & concentrate only that fraction. You might consider making modifications in your affinity purification protocol to get better purity.
"Viscous fingering". If the protein concentration in the applied sample is too high, then the viscosity is too high to allow sample to enter the bed evenly. If the sample is made visible with color (add blue dextran) you will see not a band migrating down the column, but rather 'fingers' of color. Broad band or
even double "band" elutes. Lower the protein concentration.
Hi,
Other chromatographic methods are required to purify the target protein before concentration.
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