Andrew J Ippolito wrote:
>> Dear readers,
> I am a very frustrated 3rd year graduate student that has been
> attempting unsuccessfully to purify a functionally active GST-fusion
> protein kinase expressed via pGEX from DH5-alpha e.coli.
It's not clear from your post whether it's an expression
problem or a purification problem. Assuming that your
protein can express well (check by SDS-PAGE), you can modify
some of the steps below (they are just suggestion, you will
need to determine if the modification is suitable for your
protein):
> 1. After transformation select a colony (or several) and prepare overnight
> culture (5 ml)
> 2. Grow 150 ml culture to OD595 to .6
> 3. Induce with 1mM IPTG for 2 hours.
> 4. Spin bacteria down for 10 minutes at 5k refridgerated.
> 5. Resuspend pellet in 1 ml of Buffer: 50mM HEPES 7.4, 5 mM EDTA, .1%
> NP-40, with .5 mM PMSF and 5 ul of aprotinin added fresh.
> 6. Freeze/thaw 3x by dry ice/37C water bath.
Lyse cell by sonication unless there is good reason why it
should not be done. Check that the cells are lysed properly
by your method.
> 7. Spin 10'x13k refridgerated.
> 8. To pellet add 500 ul 6M Guanidine:HCl in TBSt as above. Resuspend.
> 9. 15' ice.
> 10. Spin 10'x13k refridgerated. Dilute sup with 5 ml above buffer + 300 ul
> glut. beads in 1:1 slurry with above buffer.
In this step after spinning, first dilute the protein/6M
GuanidineHCL solution by pippetting with constant swirling
into 50mM Tris/1mM EDTA with added 0.5-1 M arginine (the
buffer should be cold, protease inhibitors may be added if
possible). Shake for an hours or two, or leave overnight
with shaking. If there is disulphide bonds in your protein,
redox reagents (e.g. oxidised and reduced glutathione)
should be added to the buffer.
Dilute this further 5-10X with your buffer (without
arginine). You can then purify the protein by adding GST
beads (I prefer using GST column, although I see that you
don't elute your protein from the GST bead).
> 11. 30' cold room, rocking.
> 12. Wash beads in 5-10 ml above buffer with inhibitors 10'x3 times.
> 13. Wash beads 2x in kinase buffer (25mM HEPES 7.4, MgCl2 5mM, MnCl 10mM,
> NaCl 10mM, 5% glycerol)
> 14. Assay via incubation of 10 ul beads with 10 uCi [32P]g-ATP in kinase
> buffer at 37C for 30 minutes.
>> according to the postdoc, this works. Not for me. And it is growing VERY
> frustrating! Can anyone help? I thank the list in advance for any input.
>> Sincerely,
>> Andrew Ippolito
> RPCI
>> we have tried denaturing and nondenaturing conditions (she denatures with 6
> M guanidine:HCl in Tris-buffered saline w/ .5% tween20)
