248. Purification of the protein
Aim: Purifying the protein C2 produced by BL21(DE3) using a Fast Purification Liquid Chromatography.
Protocol: follow in this link
What we did in the lab:
Materials:
• Fast Protein Liquid Chromatography
• Chaotropic reagent (Guanidinium HCL 6M)
• EDTA 0.1 M
• PMSF (100 mM)
• Ni2+ solution (100mM)
• Centrifuge
• Buffer A ( Tris 50 mM pH 7.4, NaCl 150 mM)
• Buffer B ( Tris 50 mM pH 7.4, NaCl 150 mM, Imidazole 250 mM)
Method:
Melt the pellet of bacteria C2 (from 1 l culture) and resuspend it with 10 ml of buffer A. We had 25 ml of pellet we complete until 40 ml with buffer A.
Add 40 µl of PMSF 100 mM to avoid protein denaturation.
Put the column on the FPLC, clean the FPLC with water and then fill it with buffer A.
Sonicate the sample three times one minute at 60 %, wait 90 seconds between each sonication.
Centrifuge 25 min at 16000g (rotor JA 25.50)
Inject your sample in the FPLC
Get back several samples:
• C: Crude extract : before centrigugation
• P: Pellet
• SN: Supernatant
• F: Flow through (unfixed proteins)
• W: Wash 5 % of buffer B
• Fractions (depending on the gradient)
