Western Blot Recipes

Western Blot Lower Gel Buffer (WB-LGB)

 

1 Gel

2 Gels

3 Gels

10 mL Acrylamide

20 mL Acrylamide

30 mL Acrylamide

12.4 mL ddH2O

24.8 mL ddH2O

37.2 mL ddH2O

7.5 mL LGB (4x)

15.0 mL LGB (4x)

22.5 mL UGB (4x)

10 uL TEMED

20 uL TEMED

30 uL TEMED

200 uL APS

400 uL APS

600 uL APS

 

Vortex first three ingredients, then add APS and TEMED.

 

 

APS (Ammonium Persulfate) 10% Stock

 

1 g. APS into 900 uL ddH2O (10%)

 

 

Western Blot Upper Gel Buffer (WB-UGB)

 

1 Gel

2 Gels

3 Gels

2.7 mL Acrylamide

5.4 mL Acrylamide

8.1 mL Acrylamide

12.1 mL ddH2O

24.2 mL ddH2O

36.3 mL ddH2O

5 mL UGB

10 mL UGB

15 mL UGB

10 uL TEMED

20 uL TEMED

30 uL TEMED

200 uL APS

400 uL APS

600 uL APS

 

 

12% Gel:

12 mL Acrylamide

10.4 mL ddH2O

7.5 mL LGB

 

20x TBS

48.44 g. Tris (pH 7.4)

151.9 g. NaCl

700 mL H2O

Adjust pH to 7.4

q.s. to 1 liter

 

1x TBS Blocking Buffer

50 mL 20x TBS stock

1 mL Tweene

10 mL NaN3 (Sodium Azide Stock, 0.2%)

20 g. milk powder

1 L H2O

 

TTBS

50 mL 20x TBS

1 mL Tweene

q.s. 1L H2O

 

NaN3, 0.02%, 1000x (Sodium Azide)
2 g. NaN3

100 mL ddH2O

 
10x Running Buffer
30.3g tris base (trizma)
144.1g glycine
10g SDS
q.s. to 1000ml with dd H2O
pH 8.3

1x Running Buffer
dilute 10x with ddH2O


10x Transfer Buffer

30.3g tris base (trizma)
144.1g glycine
q.s. to 1000ml with ddH2O
pH 8.3

1x Transfer Buffer

100ml 10x Transfer Buffer
500ml ddH2O
200ml methanol
q.s. to 1000ml with ddH2O 

SDS Sample Buffer, 5x

 

1.56 mL 2M Tris pH 6.8

2.5 mL BME

5.0 mL Glycerol

1.0 g. SDS

0.0005 g. Bromophenol Blue

q.s. to 10 mL w/ ddH2O

 

Tris, 2M, pH 6.8

150 mL H2O

60.55 g. Tris

Adjust to pH 6.8 w/ HCl (will take a lot… ~75-100 mL)

q.s. to 250 mL w/ H2O

 

SDS Sample Buffer Stock

15.6 mL 2M Tris (pH 6.8)

25 mL BME

50 mL Glycerol

10 g. SDS

0.005 g. Bromophenol Blue

q.s. to 100 mL w/ ddH2O

 

Antibody 12CA 519

1:500 dilution:

10 mL milk

20 uL 12CA 519

 

Antibody Rabbit Anti Mouse

1:1000 dilution:

10 mL milk

10 uL Rabbit Anti Mouse

 

1:500 dilution:

10 mL milk

20 uL Rabbit Anti Mouse

 

I-125

1:1000 dilution

30 uL I-125

30 mL milk

 

1 M CAPS, pH 11.0 (N-cyclohexyl-3-aminopropanesulfonic acid)

221.3 g. CAPS

40 g. NaOH

q.s. to 1 L

*Check pH; (should be 11.0) may need to alter slightly.

Store in dark bottle at room temperature