I am trying to purify a protein via Ni-NTA chromatography. The theoritical size of my protein is 58 kDa which after addition of his tag and thrombin site adds upto 2-3KDa resulting in protein band around 61-62 KDa. However, my protein band is always accompanied by additional band around 55 kDa with same intensity. I tried to separate the proteins via ion exchange but again the two bands are eluting together.
I am not able to identify the reason behind this.
I am attaching a gel image for reference wherein the elution fraction was of 150mM imidazole and the lysis buffer had 25 mM imidazole. I previously tried eluting the protein using smaller gradient however, the two bands always comes out together.
I am unable to figure out whether this is degradation of my protein or a contaminating band is accompanying my protein
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