Western blots

1.     Prepare protein lysates in 1x SDS sample buffer

2.     Boil lysates 5 min at 95C

3.     Run lysates on SDS-PAGE gel in 1x Laemmli running buffer

4.     Soak membrane in MeOH, then in Transfer Buffer + 10% MeOH.

5.     Transfer 1 hr at 70V at room temp or overnight at 25V at 4C in 1X Transfer Buffer +10% MeOH

6.     Block in PBST + 5% nonfat dry milk for 10 mins – 1 hour at room temp, or overnight at 4C

7.     Incubate 1hr at room temp or overnight at 4C in primary antibody diluted in PBST

8.     Wash 2 x 10 min in PBST

9.     Incubate 1hr at room temp in HRP-conjugated secondary antibody diluted 1:10,000 in PBST

10.  Wash 2 x 10 min in PBST

11.  Drain and develop using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) (incubate membrane with substrate for 5 minutes)

12.  Expose film in dark room, develop

4x SDS Sample Buffer

4mL of 100% glycerol

2.4mL of 1M Tris pH 6.8

0.8g SDS

4mg Bromophenol blue

0.5mL BME

to 10 mL w/ H2O

10X Laemmli Running Buffer

30 g Tris Base

144 g Glycine

10 g SDS

to 1L w/ H2O

10X Transfer Buffer

30 g Tris Base

144 g Glycine

to 1L with H2O

1X Transfer Buffer +MeOH

100 mL 10X Transfer Buffer

100 mL MeOH

to 1L with H20

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