13.Raf-1 Assay（和賀）
M.Aihara
previous required materials
conc stock vol
lysis buffer: Tris-HCl pH 8.0 20mM 1M 200μl for 10ml
(20ml for 10samples) NaCl 137mM 5M 274μl
Triton- X 1% 10% 1000μl
Vanadate 1mM 100mM 100μl
beta-glycerophos 10mM 1M 100μl
glycerol 10% 100% 1000μl
SDS 0.1% 10% 100μl
DTT 1mM 1M 10μl
EDTA 2mM 500mM 40μl
PMSF 1mM 100mM 100μl
aprotinin 10μg/ml 3.3mg/ml 30μl
H2O 7.046ml
washing buffer T-H pH 7.6 100mM & LiCl 0.5M
kinase buffer T-H pH 7.6 20mM & EGTA 2mM & MgCl2 10mM
Protein A sepharose(pharmacia)
anti-Raf1 antibody (Santacruz)
10% SDS-PAGE gel
loading buffer 5×
low molecular weight marker (Amersham)
Protocol (sample:CHO cells cultured on 10cm dish)
1. ligand stimulated CHO cell culture plate ( 10cm dish) was homogenized in 500μl of lysis buffer. As a control, use lysis buffer only!!
rotate 10' 4℃
centrifuge 15000rpm 10'
2. 500μl of supernatant was mixed with 50μl of ProteinA sepharose slurry (1:1) previously equilibrated with lysis buffer(preclear for non-specific binding to Protein A)
incubate in 4℃10'
centrifuge 3000rpm
3. take 400μl of sup. and replace another tube with 20μl of anti-Raf1 antibody and 70μl of protein A sepharose slurry.
rotate at 4℃ 1.5-2hr note:don't rotate over 2 hours!!
centrifuge 3000rpm
4. wash precipitate with ; lysis buffer 2 times
T-H pH 7.6 100mM & LiCl 0.5M 2 times
kinase buffer 1 times
note:It is favorable to change eppendorf tubes in each steps!
5. add 100μl of kinase buffer and take 80μl of slurry to another tube
6. aspirate buffer (about 15μl of beads will be left)
His-tagged MEK 1.5μg
GST KN-MAPK 2.5μg
cold ATP 100μM
32P ATP 10μCi (1μl)
25μl of kinase buffer / 1 tube
orders of mixing:
1.preparation of MEK(stock 50μl) in kinase buffer
for 24 samples: MEK 36μg + kinase buffer 180μl
2.preparation of MAPK(stock 25μl) in kinase buffer
for 24 samples: MAPK 60 μg +kinase buffer 160μl
3.preparation of mixture of cold & hot ATP(100μM+10μCi/ tube) in kinase buffer
for 24 sample: ATP (10mM) 7μl+32P-ATP 25μl (10μCi/tube)+kinase buffer 180μl
4.at first add 8μl of No.1 solution and spin down
5.add 8μl of No.2 and 3 solution respectively and spin down and shake gently
note: use new tube of MAPK and MEK!
incubate 30℃ 30' (shake slightly on the way of incubation)
7. stop reactions with 7μl of Laemli buffer 5×
8. shake and boil for 5' in 100℃
9. subject on SDS-PAGE (10% hand made gel,double loading lack 40mA c.c. 2hrs)
10. CBB staining and wash
11. gel dry
12. Fuji BAS imaging (2 or 3 hours)
** If you can do well, 2 hot bands are appeared, upper is GST-KN MAPK(70k) and lower is His-MEK(50k).
control tube
nega con. low molecular weight marker
raf-1 IP ＋ － －
His-MEK 1.5μg 1.5μg 1.5μg
GST-KN-MAPK 2.5μg 2.5μg