PP2C assay_生物行

Thursday, December 04, 2003

Description
PP2C assay

Procedure
1. Phosphorylation of casein
Casein kinaseI (NEB), 1000U/ul
Sigma casein C-4765, 5% solution (56mg/ml), dilute to 10mg/ml
500ul reaction, 30oC, 4h
50 mM Tris-HCl, pH 7.5
10 mM MgCl2
5mM DTT
2.5 mg casein (partially dephosphorylated) 5mg/ml
3000U CKI (3ul)
500 uCi gamma-32P-ATP (3000Ci/mmol)

To measure the incorporation efficiency, take 1 ul, add 100 ul 20% TCA, spin,
count the supernatant with 4 ml SCF (compare with 1 ul + 4 ml SCF, 100%)
Load onto a Centricon-10 tube, spin at 6Krpm at 15oC for 45 min
Add 10 vol of cold 20% TCA, keep on ice for 10 min, spin 1min
rinse with 200 ul 20% TCA, spin, remove all TCA
resuspend in 500 ul of 200mM Tris, pH7.5/1mM EDTA
(rocker platform for2h at 4oC)
check the pH, the cpm (106 cpm/ul/5ug per reaction)

2. Protoplast Lysis Buffer (PLB)
20mM TRis-HCl, pH 7.5
20mM KCl
1mM EDTA
1mM EGTA
10 mM DTT
0.5 % Triton X-100
50% glycerol
add before cell lysis 10 ug/ml antipain, leupeptin, pepstatin, 1 mM PNSF (100mM stock in isopropanol, stored at -80oC).
Add 1 mM NaF, 1 mM NaVO3 for kinase assay
add 50-100 ul PLB/ 105 protoplasts, incubate on ice for 3 min, spin 1 min. (stored at -80oC). Do not spin if membrane protein is going to be analyzed.

3. PP2C reaction (casien phosphatase)
Use 2-10 ul maize protoplast extract, 25-50 ul reaction, 30oC, 30 min, chill on ice
Use 1-2 ul E. coli extract, 25 ul reaction, 30oC, 15 min, chill on ice
50 mM Tris-HCl, pH 7.5
10 mM MgCl2 (5 mM is better, 11/2/96)
5mM DTT
Take 20 ul, add 200 ul cold 20% TCA for plant extract (take 10 ul, add 100 ul cold 20% TCA for E. coli extract), inc 5 min, spin 1 min, count 180ul (90ul) + 3ml SCF
replace MgCl2 with
a. 5 mM EDTA
b. 5 mM EGTA + 10 mM MgCl2
c. 1, 5, 10 mM CaCl2 + 10 mM MgCl2
d. check CaCl2 conc. (0. 0.1, 1, 10, 100, 1000 uM, 10mM)
Add OKA 1 uM, cyclosporin A 10 uM (or 5 mM EGTA) to inhibit PP1, PP2A, PP2B, add 100 uM H89, 100 nM K252a, staurosporine to inhibit CKI.

4. Casein kinase activity
like 1 except use cell extracts
50 ul reaction (Tris, DTT, MgCl2, or replace Mg with CaCl2)
10 ul cell extract

5. PP2C assay using E. coli cell extracts
like 2 & 3 except use E. coli cell extracts (without & with IPTG induction)
3ml cells, resuspend in 100 ul, dilte 1 ul to 100 ul, use 1-2 ul /25 ul assay, 30oC, 15 min
10 mM MgCl2, 50 mM Tris-HCl, pH 7.5, 5 mM DTT
replace MgCl2 with
5 mM EDTA
5 mM EGTA + 10 mM MgCl2
1, 5, 10 mM CaCl2 + 10 mM MgCl2

6. E. coli cell extracts
clone PP2C to pET19b, check miniprep DNA, store DNA at -20oC
make competent BL21 cells
(grow 20ml culture in LB to OD 0.9, chill cells in ice water, spin in a 50 ml tube at 4oC at 3Krpm for 10 min. resuspend cells using a loop, add 2 ml TfbI, incubate on ice for 30 min, spin in a 15 ml tube at 3Krpm for 10 min. resuspend in 1 ml TfbII, make 50 ul aliquots, freeze at -80oC)
add 0.5 ul miniprep DNA into 10 ul competent BL21 cells
incubate on ice for 20 min
heat shock at 37oC for 2 min
add 100 ul LB, grow at 37oC for 1 h
plate 20-50 ul on LB plates (100 ug Amp + 30 ug Cam)
incubate at 37oC for 12 h
pick a few colonies, inoculate 10 ml culture in LB (Amp + Cam) (5 ml / tube)
grow cells to OD 0.8 (2h at 37oC)
add IPTG (100 mM stock in H2O, freeze at -20oC) to 0.5 mM
grow for 2 h at 37oC
chill cells on ice
spin down cells in a 15 ml tube at 3 Krpm for 10 min
resuspend and wash cells in 1 ml of cold 0.3 M mannitol, 20 mM Tris-HCl, pH 7.5, 4 mM KCl
divide cells to 3 tubes 300 ul each, spin for 1 min at 4oC
remove supernatnat and freeze cell pellets
thaw cell pellets
add 200 ul PLB (plus 5 mM spemidine to pellet DNA-protein complex), incubate on ice for 5 min, spin for 30 min at 4oC, load gel or perform PP2C assay using the cell extracts (store at -80oC). stable for several months (4 months so far).

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PP2C assay