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Prep of 6-His tagged Protein from BL21

Lysis Buffer: 20 mM Tris pH 7.9, 500mM NaCl, 5 mM Imidizole, 0.5 mg/ml Lysozyme, 0.5% NP-40, 1mM PMSF, LPC, DTT or Beta-Mercaptoethanol

Wash Buffer:  20 mM Tris pH 7.9, 500mM NaCl, 30mM Imidizole, 0.5% NP-40

Elution Buffer:  20 mM Tris pH 7.9, 200mM NaCl, 300mM Imidizole

 

1.   Start a 50-100 ml overnight culture of a BL21 strain transfected with a construct coding for a 6-His tagged protein.  Include the appropriate antibiotics. Grow overnight at 37 degrees C.

2.   In the morning, inocluate 1 liter of 2xYT media (rich media) with 10-20 ml of the saturated overnight culture, again using the appropriate antibiotics.  Grow at 37 degrees C. 

3.   When the culture has an OD600 between 0.4 and 0.8 (2-6 hours, check periodically) induce expression of the protein using IPTG (in the case of pET vectors).  For induction, remove the culture growing from 37 degrees C and place in an ice water bath until cooled and then add IPTG.  For a 3 hour induction use 1 mM IPTG, for longer inductions use less IPTG.   If cooling the shaker during this step, remember to leave the door open and to turn off the heater.

4.   Return the culture to the incubator.  If inducing a Xenopus protein it is advisable to set the incubator at 20 degrees C for induction (more physiological temperature).

5.   Spin down the pellets after a few hours and place in the -80 deg C freezer overnight.

6.   Thaw the pellet on ice and resuspend with a dounce homogenizer in ice cold Lysis Buffer.

7.   Incubate the resuspended cell pellet on ice for 15-20 minutes to allow the lysozyme to work.

8.   Sonicate for 2-5 minutes, be sure to keep the lysate cool while sonicating.  Sonication can heat a sample so periodically swirl the sample and turn of the sonicator.

9.   After the solution has thin out a bit, spin in an Oakridge tube for ~45-60 minutes at 17,500k at 4 degrees C.

10.            Take the supernatant and add to 1-2 ml of washed Ni-NTA agarose beads.  Rotate at 4 degrees C for at least one hour in a 50 ml conical.  If performing a insoluble prep as well see the steps listed below.

11.            Wash beads 3-4 times with ice cold wash buffer.

12.            Elute 4 times with ice cold elution buffer and place elutions on ice. 

13.            Check elutions by Bradford in a 96 well dish with 150 ul of 1x Bradford and 2 ul of protein per well.

14.            Run a coomassie gel of samples taken throughout the prep to measure the purity of and recovery of your sample.  Combine appropriate fractions.  Freeze in lN2.

 

Preparing an insoluble pellet

1.   Resuspend the insoluble pellet (step 9 from above) in room temp. Lysis buffer + 6m Guanadine HCl using RT dounce homogenizer.  Be sure not to do this on ice, as the salt will crash out of solution quickly if placed on ice. 

2.   Spin in Oakridge tubes at 17,500k for 30-45 minutes at 22 deg C.

3.   Take the supernatant and incubate with 1-2ml's of washed Ni-NTA agarose beads for a minimum of 1 hour at room temperature.

4.   Wash the beads 2 times with RT Wash buffer + 6M Guanadine Hcl (no detergent).

5.   Wash the beasds 2 times with RT Wash buffer + 6M Urea.

6.   Elute the beads in RT Elution buffer + 6M Urea. Keep elutions at room temperature and check by Bradford for protein.

7.   Run a coomassie gel of samples taken throughout the prep to measure the purity of and recovery of your sample.  Combine appropriate fractions.  Freeze in lN2.

 

 

This page last modified 08/11/2011

The Stukenberg Lab and the Burke Lab are in the Department of Biochemistry and Molecular Genetics at the University of Virginia