Protocols

YRS Protein Expression & Purification

Buffer Recipes:

5x Ni-NTA Binding Buffer*:

50%                        Glycerol

2.5 M                       NaCl

75 mM                    Imidazole

100 mM                 Tris-HCl - pH 7.9

 

5x Ni-NTA Wash/Elution Buffer Base*:

2.5 M                       NaCl

100 mM                 Tris-HCl – pH 7.9

 

1x Ni-NTA Binding Buffer:

20%                          5x Ni-NTA Binding Buffer

5 mM                       BME**  

 

1x Ni-NTA Washing Buffer:

20%                          5x Ni-NTA Wash/Elution Buffer

40 mM                    Imidazole

5 mM                       BME**

 

1x Ni-NTA Elution Buffer:

20%                          5x Ni-NTA Wash/Elution Buffer

275 mM                 Imidazole

5 mM                       BME**

 *Filtered through 0.22 um filter and stored in the cold room

**BME should always be freshly added… BME loses its reducing capacity after a few days

 

Protein Expression in E coli

1. 1. Start with a freshly transformed plate of BL21 cells containing your expression vector
   2. Prepare your LB in the autoclave and cool sterilized LB to ice cold temperatures
      1. 3L of LB in the 6L flasks work best for this protocol. Anything smaller will result in overgrowth.
      2. Add the proper antibiotic and drop a single bacterial colony into the 3 liters of LB using the sterile technique.
      3. Put flasks on the 37 degree shaker for roughly 14 hours
      4. Measure OD₆₀₀ periodically and wait for an OD₆₀₀ of ~0.8
      5. The flasks are now ready to come off the shaker
      6. Give the cultures a cold shock in an ice bath for 30 minutes
      7. Induce cultures with IPTG for a final IPTG concentration of 0.5 mM
      8. Put flasks on the room temperature shaker for 5-6 hours
      9. After 5-6 hours, begin collecting E coli cultures in the J6 centrifuge
      10. 20 minute spins at 4300 rpm will do the trick
      11. Decant liquid and store the E coli pellets
          1. Pellets can be resuspended immediately in 1X NTA binding buffer(~150 mL total buffer for an 18 L pellet)  and then stored in the -20 degree freezer or pellets can be put directly into the -20 degree freezer

 

Protein Purification of 6x His tagged proteins (Based on purifying a 18 L culture)

1. 1. Thaw pellets and add 1x NTA binding buffer (if not already resuspended)
      1. Either add the binding buffer (~150 mL total buffer for the entire 18 L pellet) and put the bottles on the shaker or
      2. Thaw the resuspended culture in cool water and put on the shaker
      3. Add BME to this thawed and resuspended E coli mixture
      4. Lyse cells using the Microfluidizer
      5. Spin E coli lysate in the ultracentrifuge at 35K rpm for 30 minutes
      6. Take the spin time as an opportunity to prepare your Ni-NTA column

                                               i.     Add your desired amount of resin to a clean column

                                             ii.     Allow resin to settle and flow storage buffer out of the column

                                            iii.     Calibrate the resin bed with at least 1.5X CVs of binding buffer

1. 1. Collect supernatant after the spin and dilute to 1 L
   2. Pass this diluted mixture through the calibrated Ni-NTA column (multiple times through the column should result in a higher protein yield)
   3. Wash the column with ~750 mL of 1x wash buffer
   4. Elute the protein with 1.5X-2X CVs of elution buffer
   5. Buffer exchange protein (dialysis or desalting column) into a more suitable buffer (e.g. PBS + 5mM BME) for further purification or storage.

 

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