Raf 1 assay


previous required materials
conc stock vol
lysis buffer: Tris-HCl pH 8.0 20mM 1M 200l for 10ml
(20ml for 10samples) NaCl 137mM 5M 274l
Triton- X 1% 10% 1000l
Vanadate 1mM 100mM 100l
beta-glycerophos 10mM 1M 100l
glycerol 10% 100% 1000l
SDS 0.1% 10% 100l
DTT 1mM 1M 10l
EDTA 2mM 500mM 40l
PMSF 1mM 100mM 100l
aprotinin 10g/ml 3.3mg/ml 30l
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 500l of lysis buffer. As a control, use lysis buffer only!!
rotate 10' 4
centrifuge @15000rpm 10'
2. 500l of supernatant was mixed with 50l of ProteinA sepharose slurry (1:1) previously equilibrated with lysis buffer(preclear for non-specific binding to Protein A)

incubate in 410'
centrifuge 3000rpm
3. take 400l of sup. and replace another tube with 20l of anti-Raf1 antibody and 70l 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 100l of kinase buffer and take 80l of slurry to another tube
6. aspirate buffer (about 15l of beads will be left)

His-tagged MEK 1.5g
cold ATP 100M
32P ATP 10Ci (1l)
25l of kinase buffer / 1 tube
orders of mixing:
1.preparation of MEK(stock 50l) in kinase buffer
for 24 samples: MEK 36g + kinase buffer 180l
2.preparation of MAPK(stock 25l) in kinase buffer
for 24 samples: MAPK 60 g +kinase buffer 160l
3.preparation of mixture of cold & hot ATP(100M+10Ci/ tube) in kinase buffer
for 24 sample: ATP (10mM) 7l+32P-ATP 25l (10Ci/tube)+kinase buffer 180l
4.at first add 8l of No.1 solution and spin down
5.add 8l 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 7l 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 1.5g 1.5g 1.5g
GST-KN-MAPK 2.5g 2.5g