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. The protein has been
published as a functionally active autophosphorylating kinase which also
phosphorylates several protein substrates.
I am using identicle stocks of DNA to transform competent bacteria that
a former postdoc has used to successfully purify the protein. She has since
left our lab, and we have been unable to reproduce her protocols. I have
tackled the problem from all different sides, all to no avail. Here is the
current protocol she insists works; if anyone has any comments on it, I will
appreciate it greatly :)
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.
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.
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)
