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Back to Eccles Lab > DGG Protocols

Contents

MITF western blot protocol (Zymed C5+D5 antibody)

Total cell Lysate

Method KEEP ALL REAGENTS ON ICE TO USE

RIPA buffer

(without protease inhibitors) store at 4°C

1 M Tris.Cl pH8.0 0.5ml 0.05M
1 M NaCl 1.5ml 0.15M
10% NP-40 1ml 1%
Sodium deoxycholate 50mg 0.5%
10% SDS 100ul 0.1%
MQ H20 6.9ml
Total 10ml

Cell lysis buffer

(only prepare what you need)

20X Complete protease inhibitor 50ul
0.1 M PMSF (available in freezer) 10ul
100 uM NaOrthovanodate (available in freezer) 10ul
RIPA buffer 930ul
Total 1000ul

PAGE Gels

TG X10

15.14g Tris (0.25M)
72g Glycine (1.9M)
Make up to 0.5L with dH20


TGS

100ml TGx10
5ml 10% SDS
895ml dH20

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 dH20



Western Transfer

1x Blot Buffer

100ml TGx10
200ml 100% methanol
10ml 10% SDS
690ml dH2O

Immuno

Use Western Breeze kit & follow their instructions.