In vivo Kinase Assay
Name
Pierce protein G magnetic
beads
Brand
Cat. No
Thermo Scintific
88848
Anti Flag M2
Sigma
F11804/SL11063
IRE1α (14C10) Rabbit mAb
Cell Signaling
3294S
Thiophosphate Ester
Abcam
ab133473 (FOR IP)
Rbm Ab Thiophosphate
Abcam
ab92570 (FOR BLOT)
C-Myc
Santa Cruz
SC-40
Digitonin
p-Nitrobenzyl mesylate
(PNBM)
Sigma
D141
N6-Furfuryl-ATP-Gamma-S
Abcam
Biolog Life Science
ab138910
F008
( 6-cPe-ATP-γ-S )
Biolog Life Science
p044-25
ATP Disodium Salt
Santa Cruz
sc-20240A
ATP Gamma S
Abcam
ab138911
 Grow 10X150 cm2 dishes for each cell type used.
 Do PEI transfection to HEK293 cells with WT and mutant of your kinase. (1ug DNA/3ul
PEI).
 At the same day with transfection do antibody conjugation (use 15ul magnetic bead/ IP
sample, and 4ug antibody)
 Do not forget to block beads for 1 hr at +4 oC with 500ul %2 PBS-BSA in 1.5 ml
eppendorfs. (If you want to block longer use %1 PBS-BSA)
 After 24 hours of PEI transfection add 300nM thapsigargin for 1 hour as an ER-stressor.
 Wash cell with PBS, collected with trypsin and centrifuge the cells, remove DMEM and
washed with PBS again. (1100rpm 4 min 4oC) (do all in 15 ml falcon)
 Then add the wash buffer with 30 µg/ml digitonin to the cells at 3X cell volume and
resuspend the cells in this buffer, transfer cells to eppendorf after dissolving in digitonin
kinase buffer mix and rotate +4 oC for 10 mins.(From 30mg/ml stock of digitonin, add
30ug/ml concentration to kinase buffer= 1:1000 dilution. This is for wash no inhibitor
cocktails added)
 Spin the cells down at 1500 rpm for 10 min at 4 oC
 Discard the supernatant and add kinase buffer at 2.5X cell volume.
 Again we spin the cells down at 1500 rpm for 10 min at 4 oC
 Add kinase buffer for performed the kinase reaction (including PMSF, Na3VO4, PIC,
Pase) containing 200uM ATP analogs (for Analog positive samples) and add 100uM
ATP, 1mM GTP, meaning do this reaction inside 250ul kinase buffer/ IP sample that you
prepared with all ingredients included.(starting 100ul pellet add 250ul, if you have
200ul pellet add 500ul kinase buffer etc. )
 Incubate at 30oC for 1 hr shaking rigorously (950 rpm at a thermo-shaker).
 Stop the kinase reaction adding 250ul, 2X Phospho lysis buffer (with 2x inhibitors)
with 20 mM EDTA.
 Then lyse the cells by 5 sec of vortexing for 10 min with 5 sec of intervals.
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Centrifuge the cell debris for 15 min at highest speed at tabletop centrifuge at 4 oC.
Take supernatant now and discard the pellet.
Add 12.5mM PNBM according to final volume.
Incubate at 24oC for 2 hrs shaking rigorously (950 rpm at a thermo-shaker)
Take 2ul of each sample to measure the protein concentration by BCA.
After procedure take out 50ul from each sample for loading to gel as total lysate control
before IP.
Complete the samples volume 500ul with phosphor lyses buffer. (total volume will be
1ml)
Use PD-10 coloumns (GE healtycares) for PNBM alkylation. (For thiophosphoester do
buffer exchange as PNBM intercalates with thiophosphate esters.)
Start procedure before 30 mins. (at the last half hour of PNBM alkylation). Wash
columns for 4 times with 3,5ml 1x PLB only (no inhibitors).
Then load lysates to columns and wait till all liquid comes out. Than change to new 50
ml falcon for each sample and elute with 3.5ml Phospholysis buffer including inhibitors
to those new falcons.
Then put the eluates from each sample to into three 2ml eppendorfs.
In the meantime take out your antibody conjugated beads, wash them with DPBs for 2
times and PLB for 2 times. Then add total 30ul bead / IP sample (because we divided
each sample into three 2 ml eppendorf, each eppendorf will contain 10ul bead) and IP
overnight at 4 oC.
Next day get rid of supernatants (you may take flow through as control for your IP),
combine 3 samples of each and wash 2 times with PLB (combine beads of each IP
sample in 1.5 ml eppendorf ) add 50ul PLB and 12.5 ul 5X SDS loading dye, boil at 95oC
for 5 minutes and load to gel (put eppendorfs to magnetic rack, wait for beads to go
away from lysate).
Load approximately 20ul sample to each well.
IRE-1 In vivo Wash Buffer (without Analogs)
Stock
Final cons.
Hepes pH 7.4
1mM
20 mM
KoAC
1M
50mM
MnCl2
1M
1mM
MgCl2
1M
1mM
Na2Mao4
1M
1mM
NaF
500mM
2mM
H2O
IRE-1 In vivo Kinase Buffer
Stock
Final cons.
ATP
10mm
100 µM
GTP
100 mM
1 mM
N6-Fu-ATP-gS 10 mM
250 µM
Hepes pH 7.4
1mM
20 mM
KoAC
1M
50mM
MnCl2
1M
1mM
MgCl2
1M
1mM
Na2Mao4
1M
1mM
NaF
500mM
2mM
H2O
** Added fresh phosphatase, protease inhibitors and ATP, GTP, N6-Fu-ATP-gS analogs.
PERK In vivo Wash Buffer (without Analogs)
Reagent
Stock
Final cons.
Hepes
1M
20mM
KCL
1M
50mM
MgCl2
1M
2mM
H2O
PERK In vivo Kinase Buffer (with Analogs)
Reagen
Stock
Final cons.
Hepes
1M
20mM
KCL
1M
50mM
MgCl2
1M
2mM
ATP
10mm
20 µM
N6-Phenil-ATP-gS
10 mM
250 µM
H20
** Added fresh phosphatase, protease inhibitors and ATP, N6-Phenil-ATP-gS analogs.
2X Phospho Lysis Buffer
Reagent
Stock
Final cons.
Hepes pH 7.4
1M
100 mM
NaCl
5M
200mM
EDTA
0,5M
20mM
NaPP
200mM
8mM
NaF
500mM
20mM
Triton 100x
100x
2X
H2O
** Added fresh phosphatase inhibitor.