Hello. I'm new to GST purification. I expressed a fusion protein: GST-'Protein X'. I can see expression in the soluble lysate upon over-expression. When purified with Glutathione Sepharose, the A280 reading of the eluent would indicate a ~0.5-0.8 mg/mL concentration yet when loading the sample, I'm not getting anywhere near that amount of protein. I blanked with elution buffer.
Additionally, the SDS-PAGE shows a faint band corresponding to the fusion protein and a predominant band around the size of GST alone (which is the same size as 'Protein X'). Does anyone know how I could get GST alone? There is a thrombin cleavage site but I didn't perform the cleavage! Any ideas would be much appreciated!