MBP蛋白纯化SmallScaleMBP.doc

Small Scale MBP-fusion Protein Purification http://wolfson.huji.ac.il/purification/TagProteinPurif/MBP_Tag_nature.html According to New England BioLabs ? Instruction Manual ? (pdf-I)? (pdf-II) Aliquot of Cell Pellet after Induction The idea is to aliquot cells after induction, and keep at? -80oC enough cell pellet samples for optimization of small scale purification procedure and further scale-up. ?Once you set up the best purification conditions at low scale, you can scale-up the procedure. Example:1)?? ?Grow 1L culture2)?? ?Induce (IPTG, salt induction, etc. etc.)3)?? ?Spin cell culture 10min 8000rpm 4oC, discharge supernatant4)?? ?Resuspend cell pellet at 4oC very gently with 100ml cold PBS buffer. Aliquot as following: ??? ?? a) 10 tubes (1.5ml plastic tubes) with 1ml suspension (it means 10ml original culture per tube); ??? ???b) 4 tubes (15ml plastic tubes) with 10ml suspension (it means 100ml original culture per tube)??? ???c) 1 tube (50ml plastic tube) with 50ml suspension (it means 500ml original culture). 5)?? ?Spin 10min 8000rpm 4oC, discharge supernatant6)?? ?Keep cell pellet at -80oC Equilibration of Amylose resin Place 200μl beads (400μl suspension) of Amylose resin in 1.5ml plastic tube. According to New England BioLabs the resin binds 3mg fusion protein per ml bed volume. Wash with 2 x 1.5ml H2O and 2x 1.5ml column buffer (washing: mix, spin 3min 3500rpm, discharge supernatant). ? Protein Extraction 1) Resuspend pellet of 40ml cell culture in 5ml lysis buffer. Suggested Lysis buffer : 200mM NaCl; 20mM TrisHCl pH 7.4; 1mM EDTA (COLUMN BUFFER) and additives ??????????????????????????????? ?Alternative buffers: MOPS, HEPES and Phosphate? buffers?around pH 7.0 and NaCl or KCl ?from 25mM to 1M. Optional additives to the lysis buffer a) 1mM PMSF and/or protease inhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 from Sigma or any other commercial cocktail) b) Dnase 100U/ml or 25-50μg/ml (SIGMA DN-25). Incubate 10min 4°C in t