Paul.xu: Created page with "== Expression of recombinant proteins
== '''Materials:'''
- LB medium
- 40% glucose
- Amp 100 mg/ml
- IPTG 100 mg/ml in PBS

'''Methods'''
1. Inoccula..."

2011-11-10T16:27:09Z

Created page with "== Expression of recombinant proteins == '''Materials:''' - LB medium - 40% glucose - Amp 100 mg/ml - IPTG 100 mg/ml in PBS '''Methods''' 1. Inoccula..."

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== Expression of recombinant proteins ==

'''Materials:''' - LB medium - 40% glucose - Amp 100 mg/ml - IPTG 100 mg/ml in PBS 

 '''Methods''' 1. Inocculate 100 ml LB medium + 1 ml glucose + Amp with 1 fresh colony 2. Grow overnight 3. Inocculate 1 l LB medium + 10 ml glucose + Amp , with 1 ml preculture 4. Grow till OD600 ~ 0.7, take a 1 ml sample 5.  Prepare fresh IPTG, add 1 ml 6. Incubate 3 hours 7. Take 1 ml sample for expression control 8. Cool culture 15-20â² on ice 9. Centrifuge 30â² at 2000 g, 4 oC 10. Pellet can be stored at -80 oC 

 '''Expression QC''' â¢ pellet the 1 ml samples and wash 2x in 1.5 ml cold PBS â¢ add100 ÎŒl dH2O and vortex â¢ add 20 ÎŒl 5x SDS-loading and boil 5â² â¢ run 12 ÎŒl on a SDS-PAGE gel â¢ stain with Coomassie

 

== Purification of inclusion bodies ==

 Stock solutions â¢ lysozym 50 mg/ml in dH2O (store at -20 C) â¢ DNase 1 mg/ml in 50% glycerol, 75 mM NaCl (store at -20 oC â¢ MgCl2 0.5 M in dH20 â¢ Tris-Cl 2.5 M pH 8.0 â¢ NaEDTA 0.5 M in 50 mM Tris-Cl, pH 8.0 â¢ NaAzide 30% in PBS â¢ Na deoxycholate 10% in dH2O â¢ Triton X-100 10% â¢ 5 M NaCl â¢ DTT 1 M

'''Buffers''' 

 '''Solution buffer''' (13 ml), pH 8.0 50 mM Tris-Cl 260 ÎŒl stock 25% sucrose 3.25 g 1 mM NaEDTA 26 ÎŒl stock 0.1% NaAzide 43.3 ÎŒl stock 10 mM DTT 130 ÎŒl stock

'''Lysis buffer''' (12.5 ml) 50 mM Tris-Cl 250 ÎŒl stock, pH 8.0 1% Triton X-100 1.25 ml stock 1% Na deoxycholate 1.25 ml stock 100 mM NaCl 250 ÎŒl stock 0.1% NaAzide 42 ÎŒl stock 10 mM DTT 125 ÎŒl stock

'''Washing buffer with Triton''' (10 ml) 50 mM Tris-Cl 200 ÎŒl stock pH 8.0 0.5% Triton X-100 0.5 ml stock 100 mM NaCl 200 ÎŒl stock 1 mM NaEDTA 20 ÎŒl stock 0.1% NaAzide 33.3 ÎŒl stock 1 mM DTT 10 ÎŒl stock 

 '''Washing buffer without Triton''' (10 ml) 50 mM Tris-Cl 200 ÎŒl stock pH 8.0 100 mM NaCl 200 ÎŒl stock 1 mM NaEDTA 20 ÎŒl stock 0.1% NaAzide 33.3 ÎŒl stock 1 mM DTT 10 ÎŒl stock

11. Resuspend pellet in 13 ml solution buffer on ice and transfer it to a 30 ml centrifugion bottle 12. Sonicate 50% level 4-5, 30 pulses, on ice 13. Add 100 ÎŒl lysozym, 250 ÎŒl DNase I, 50 ÎŒl MgCl2, vortex 14. Add 12.5 ml lysis buffer and vortex short 15. Incubate 30-60â² at RT 16. Add 350 ÎŒl NaEDTA 17. Freeze in N2 until it stops bubbeling and thaw for 30â² at 37 oC 18. Add 200 ÎŒl MgCl2 19. Wait until viscosity decreases 30-60â² 20. Add 350 ml NaEDTA

From now on everything should be done on ice 

21.  Pellet at 11,000 g, 20â², 4 oC 22.  Discard supernatant and take a sample 23. Resuspent pellet in 10 ml washing buffer with Triton 24.  Sonicate 50% level 4-5, 30 pulses, on ice 25. Pellet at 11,000 g, 20â², 4 oC 26. Discard supernatant and take a sample 27. Resuspent pellet in 10 ml washing buffer without Triton 28. Sonicate 50 % level 4-5, 30 pulses, on ice 29. Pellet at 11,000 g, 20â², 4 oC 30. Discard supernatant and take a sample

== Dissolving of recombinant protein ==

31. Dissolve pellet in 9 ml 8 M Guanidinium pH 8.0 + 4 mM DT 32. Shake at RT until pellet dissolves 33. Aliquot in 1 ml fractions and store at -80 oC

 

== Refolding ==

 '''200 ml refolding buffer''' 100 mM Tris-Cl: 8 ml stock 400 mM L-Arginine: 16.9 g 2 mM NaEDTA: 0.8 ml stock 0.5 mM ox.glutathione: 61.2 mg 5 mM red. glutathione: 308 mg protease inhibitors: 50 ÎŒl cocktail

34. Add 1 ml dissolved protein drop by drop using a 1 ml syringe and a # 27 meedle to the refolding buffer while vigorously stirring. 

35. Stir slow, 8 hours at 4 oC 

36. Again add 1 ml 

37. Stir slow, 8 hours at 4 oC. 

38. Again add 1 ml 39. Stir slow, 8 hours at 4 oC 40. Filter through a 2 um filter and concentrate till ~ 12 ml in a 200 ml concentrator 41. Wash concentrator with 1 ml flow through and add this to the protein sample 42. Measure OD280, it should be at least 3x higher than flow through 

== Size exclusion(FPLC) ==

 Elution buffer 150 mM NaCl 20 mM Tris-Cl pH 8.0 (2um filtered) 43. Filter the protein sample through a 0.2 um filter 44. Bring 13 ml protein on the size exclusion column (hiload 26/60 Superdex 200 prep grade) using a superloop 45. Elute with the elution buffer 1ml/min, start collection 4 ml fractions from 70 ml-300 ml (unfolded protein comes of in the void volume) 46. Determine amount of protein in different fractions and run fractions on gel to check the purity of the protein

Protein Purification from Inclusion Bodies Protocol - Revision history

Paul.xu: Created page with "== Expression of recombinant proteins == '''Materials:'''- LB medium- 40% glucose- Amp 100 mg/ml- IPTG 100 mg/ml in PBS '''Methods'''1. Inoccula..."

Paul.xu: Created page with "== Expression of recombinant proteins
== '''Materials:'''
- LB medium
- 40% glucose
- Amp 100 mg/ml
- IPTG 100 mg/ml in PBS

'''Methods'''
1. Inoccula..."