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Contents

Western Blotting

SDS-PAGE

TG

15.14g Tris (0.25M)
72g Glycine (1.9M)
Make up to 0.5L with dH2O

TGS

100ml TG
5ml 10% SDS
895ml dH2O

Resolving Gel

Reagents 8% 10% 12%
37.5:1 bis/acrylamide (30%) 2.67ml3.33ml4ml
1.5M Tris HCl pH 8.8 2.5ml2.5ml2.5ml
10% SDS 100uL100uL100uL
MQH2O 4.62mL3.96mL3.29mL
10% APS 100uL100uL100uL
TEMED 10uL10uL10uL


Stacking Gel

Reagents 5%
37.5:1 bis/acrylamide (30%) 1.67ml
0.5M Tris HCl pH 6.8 2.5mL
10% SDS 100uL
MQH2O 5.62mL
10% APS 100uL
TEMED 10uL


5x Loading Dye

4mL 1M Tris pH 6.8 (0.4M)
5mL Glycerol, 100% (50%)
1g SDS (10%)
0.025g Bromophenol blue (0.25% w/v)
0.30g DTT (3% w/v)
1mL dH2O



Western Transfer

1x Blot Buffer

100mL TG
100mL 100% methanol
2.5mL 10% SDS
797.5ML dH2O

For semi-dry transfer unit 100mL of transfer buffer is plenty.

10mL TG
10mL 100% methanol
0.25mL 10% SDS
79.8mL dH2O


Immunostaining

10x TBS

24.2g Tris
80.6g NaCl
900mL dH2O
pH to 7.6 with HCl

TBST

100mL 10x TBS
1mL Tween 20
899mL dH2O

5% LFM in TBST

2.5g low fat milk powder
Make up to 50mL with TBST

0.1% LFM in TBST

0.4mL 5% LFM in TBST
19.6mL TBST