Editing HRV 3C Expression and Purification (section)

=== For GST-3C  ===

<br>9. Load the supernatant to a 5-ml GSTrap column equilibrated with wash buffer.<br>10. Wash the column with wash buffer until no more protein elutes (monitored by the absorbance at 280 nm).<br>11. Elute the protein with elution buffer. Collect the eluate in 2-ml fractions.<br> 

<br>12. Pool the protein containing fractions and dialyze overnight against 50 ml storage buffer at 4°C.<br> 

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13. For product: dilute the final solution with storage buffer to a final concentration of 1 mg/ml and store in 0.5-ml aliquots at -80°C.<br> 

<br>The amount of His6-3C protease can be determined by measuring the absorbance at 280 nm of the protein solution against the dialysis buffer. The concentration can be calculated using a specific extinction coefficient of 0.253 (a His6-3C protease solution of 1 mg/ml gives an A280 of 0.253). The molecular weight of His6-3C protease is 21.7 kDa.<br> 

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The amount of GST-3C protease can be determined by measuring the absorbance at 280 nm of the protein solution against the dialysis buffer. The concentration can be calculated using a specific extinction coefficient of 1.005 (a His6-3C protease solution of 1 mg/ml gives an A280 of 1.005). The molecular weight of His6-3C protease is 46.4 kDa.<br>