Western Blotting reagents

                                                    Western Blotting reagents

Protein Lysis Buffer „TENTE“

1x concentration:

20mM Tris (pH 7.4)

150mM NaCl

1mM EDTA

1mM EGTA

1% Triton

Add fresh: Phosphatase/protease inhibitors :

Complete Mini Inhibitor tablet (1 tablet/10ml lysis buffer)

1x PPi (see below for 50x stock solution)

50x PPi (20ml)  

		1x Final in TENTE	stock
Sodium pyrophosphate (125mM, FW 446.06)	1.11g	2.5mM
ß-Glycerophosphate (50mM, FW 216.00))	0.216g	1mM	1M	1 ml
Na3VO4 (sodium-orthovanadate) (50mM)	0.184g	1mM	0.1M	10 ml

Freeze PPI at -20 in 400µl aliquots, add 20µl per 1ml lysis buffer.

Sodium pyrophosphate: MP Biomedicals, Cat # 152579

Sodium orthovanadate: sigma S6508

ß-Glycerophosphate disodium salt hydrate: sigma G9422

5x stock TENTE (store at 4°C) ( Make 200ml )

1M Tris (pH 7.4)                                20ml

5M NaCl                                               30ml

0.2M EDTA (pH8.0)                          5ml                         (or 2ml of 0.5M EDTA pH 8.0)

EGTA (MW 380)                                0.38

Triton X-100                                        10ml

Sample loading buffer (SLB), store at RT

50ml 4x SLB

20% SDS                                               20ml

Glycerol                                               20ml (add LAST)

1M Tris pH 6.8                                    10ml

Bromophenol blue                          around 0.1g (don’t use too much)

For 4x working stock (always prepare fresh):

To 1ml add 160µl of ß-Mercapto, you can store the working stock at 4°C for up to 1 months

10x Gel Running Buffer (to prepare 4L), store at RT

121.2g Tris Base

577g Glycine

40g SDS

Fill 4 l beaker with 2 liters of milli-Q water and add reagents one at a time

Slowly add some more milli-Q water to help dissolve

Bring to 4 liter volume

Aliquot in four 1 liter bottles

10x Transfer Buffer (to prepare 4 l), store at RT

121.16g Tris Base

576.52g Glycine

Fill a 4 liter beaker with 2 liters of HOT milli-Q water (does not have to be boiling)

Add reatents one at a time

Slowly add additional milliQ water to help dissolve

Bring to 4 liter volume

Does not get autoclaved or filtered

1x Transfer buffer:

100ml of 10x Transfer Buffer

200ml MeOH

700ml ddH20

PBST: 0.1% Tween 20 in PBS (add 1ml Tween 20 to 1l PBS. Cut off tip of pipette tip before pipetting Tween)

Tris-Bufferd saline with Tween 20

20mM Tris, pH 7.5

150mM NaCl

0.1% Tween 20

(Make 10xTBS 1liter)

Blocking solution :

3% BSA in TBST

5% dry milk in TBST

Ladder :

Protin prestained ladder (-20 cell culture)