SDS-PAGE and Western Blot Recipes

2X Sample Buffer

Want

Reagent

[stock]/FW

For 20mL

125 mM

Phosphate Buffer pH 7.0

1M

2.5 mL

200 mM

DTT

 

0.31 g

20%

Glycerol (Anal-R)

 

4 mL

4% SDS

SDS

10%

8 mL

0.02%

Bromophenol Blue

 

A few crystals

 

ddH2O

 

Fill to 20mL

10X Running Buffer

Want

Reagent

For 2L

250mM

Tris Base

60g

2M

Glycine

280g

 

SDS

20g

- fill to 2L with ddH2O

5X Transfer Buffer

Want

Reagent

For 2L

250mM

Tris (not pHd)

60.6g

2M

Glycine

300.3g

-          fill to 2L with ddH2O

-          to use, dilute in 20% Methanol (in ddH2O)

Blocking Buffer (Buffer A)

Buffer B + 3%w/v skim milk powder

5X Washing Buffer (Buffer B)

Want

Reagent

For 1L

250mM

Tris-HCl pH 7.5

30.23g

750mM

NaCl

43.33g

0.25%

Tween-20

2.5mL

0.2%

NaN3

2g

-          fill to 1L with ddH2O, pH the solution with HCl

-          Buffer A is this plus 3% skim milk powder

Staining Buffer for APase (NBT/BCIP method, Buffer C)

Want

Reagent

[stock]/FW

For 500 mL

100mM

Tris-HCl pH 9.5

 

6.055g

100mM

NaCl

 

2.022g

2mM

MgCl2

3M

0.5mL

0.05%

NP40 or Tween-20

 

0.25mL

To use: 33uL 50mg/ml BCIP (in 100% DMF) + 66uL 50mg/ml NBT (in 70% DMF) + 10mL Buffer C


Resolving Gel

Target Acrylamide Concentration

7.5%

9%

10%

10.5%

11%

12%

ddH2O

4.8 mL

4.35 mL

4.0 mL

3.9 mL

3.7 mL

3.4 mL

30% Acrylamide

2.5 mL

3.0 mL

3.3 mL

3.5 mL

3.7 mL

4.0 mL

1.5M Tris-HCl (pH8.8)

2.5 mL

2.5 mL

2.5 mL

2.5 mL

2.5 mL

2.5 mL

10% SDS

100 mL

100 mL

100 mL

100 mL

100 mL

100 mL

10% APS

50 mL

50 mL

50 mL

50 mL

50 mL

50 mL

TEMED

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

*Add APS and TEMED last after degassing.

Stacking Gel

Acrylamide

4%

ddH2O

7.35 mL

30% Acrylamide

1.3 mL

1M Tris-HCl (pH6.8)

1.25 mL

10% SDS

100 mL

10% APS

50 mL

TEMED

10 mL