Phosphorylation Assay

Materials required:

Cells

PBS

HAM F-12 media

Growth factor (VEGF)

Lysis Buffer mix (w/o PMSF and Aprotinin, keep at 4°C, add PMSF and

Aprotinin right before use):

1% Triton-X100

10mM Tris

1mM EDTA

150mM NaCl

30mM PPiNa

50mM NaFi

2.1mM Na3VO4

Lysis Buffer (Final): ìl

PMSF 30

Aprotinin 6

Buffer 5964

Total 6000

Preincubation buffer: Serum-free media (30ml) + Na3VO4 (15ìl)

Wash buffer: 1x PBS (15ml) + Na3VO4 (15ìl)

Method:

1- plate cells in 12-well (or 240well) plate

2- cells in serum-free media for o/n

3- cells in preincubation media, 37°C, 5min

4- aspirate media

5- treat cells with growth factor, 37°C, 5min

6- aspirate growth factor

7- wash cells with cold wash buffer, 2x

8- add 200-300ìl lysis buffer to each well, rocking plate, 4°C, 15min

9- collect cells in eppendorf tubes, rocking, 4°C, 15min

10- spin, 14,000rpm, 4°C, 1h

11- collect supernatant, store at -80°C or go ahead with next step

12- for phophotyrosine western analysis, use 15-60ìl of lysate

13- 10 or 12 % SDS-PAGE gel

14- Blocking, 3% BSA/TBST, RT, 1h

15- 1° Ab, anti-pTyr Ab (4G10, Upstate) in blocking buffer, 4C, ON

16- wash with TBST

17- 2°Ab, RT, 1h

18- wash with TBST, at least 2h, change buffer as often as possible

19- rinse blot with dH2O, 5x

20-ECL, 2min, RT

21-Develop

mRIPA Lysis Buffer (this also works well, recipe from Tom

Graeber):

Stock 50 mls

50 mM Tris pH 7.4 1M 1:20 2.5 ml

1% NP-40 10% 1:10 5 ml

0.25% Na deoxycolate 2.5% 1:10 5 ml

1 mM EDTA 0.5 M 1:500 0.1 ml (100 ul)

0.15 M NaCl 5 M 1:33 1.5 ml

1 mM Na vanadate 100 mM 1:100 0.5 ml (500 ul)

10 mM ß-glycerophosphate 1 M 1:100 0.5 ml (500 ul)

1 mM NaF (optional)

H2O 34.9 ml

---------

50 ml

add fresh (5-10 min. prior is okay, PMSF has short half-life in aqueous

solution):

1 mM PMSF 100 mM 1:100

20 ug/ml leupeptin 10 mg/ml 1:500

20 ug/ml aprotinin 2 mg/ml 1:100

(Author：Lee, Sunyoung)