A cDNA encoding full length human UCHL1 was inserted into a eukaryotic expression vector with a C-terminal His-Tag and subsequently transformed into E. coli BL21 cells. Cells were grown in LB medium supplemented with ampicillin at 37°C. Cells were harvested after induced protein expression for 6 hrs. Soluble protein was purified using immobilized metal affinity chromatography. Yields of soluble UCHL1 protein were estimated by performing SDS-PAGE and staining with Coomassie Blue R250. Purified protein was concentrated following extensive dialysis in PBS to 1 mg/mL, and stored at -70°C (recommended for long term storage). For the subsequent analyses, the concentration of UCHL1 was obtained using the Bradford method and confirmed with a densitometry-based ImageJ quantification.
Image: His-tagged-UCHL1, was expressed and purified from E. coli BL21 using immobilized metal affinity chromatography. Protein concentration was determined using BSA Protein Bradford, and confirmed with a densitometry-based ImagJ quantification using BSA as standard. Picture shows the Coomassie gel of UCHL1 and BSA at serial dilution. UCHL1 runs at ~37 kDa.