Western Reagents

 

Towbin Buffer (5 l)

25 mM Tris-Base 15.1g

192 mM glycine 72.1 g

20% methanol  1 l

make to 5 l with ddH2O

do not adjust the pH

Towbin buffer is the same as SDS running buffer, except it contains methanol and lacks SDS.

Nitrocellulose - Schleicher & Schuell BA-S 85 supported nitrocellulose 0.45 micron

Blotting Paper - Schleicher & Schuell Blot Block 32-48940 15 x 21 cm sheets

PBS-Tween

10 mM NaH2PO4  1.38 g/l

150 mM NaCl  8.8 g/l

adjust to pH 7.2 and then add 0.3% Tween 20 (3 ml/l)

pH 9.5 Buffer

100 mM Tris-Base 6 g/500 ml

100 mM NaCl  2.9 g/500 ml

5 mM MgCl2  0.5 g/500 ml

adjust pH to 9.5

nBT-BCIP Alkaline Phosphatase Color Development Reagent

BCIP - 17 mg 5-bromo-4-chloro-3-indoyl phosphate/ ml of DMSO

store at room temp in the dark (wrapped in foil)

Sigma B8503

nBT - 33 mg nitroblue tetrazolium/ml of 70% DMSO

store at room temp in the dark (wrapped in foil)

Sigma N6876

mix the two reagents in pH 9.5 buffer just before use at the following ratio:

10 ml of pH 9.5 buffer

100 ul nBT

100 ul BCIP

0.5 M EDTA (disodium salt) 16.8 g/100 ml

antimouse IgG (whole molecule) alkaline phosphatase conjugate (Sigma A5153) or other appropriate secondary antibody

Ponceau S Stain

0.1 g Ponceau S

5 ml acetic acid

95 ml ddH2O

Western Transfer - traditional tank method

while running the gel, place transfer sandwich and sponges in a tray containing Towbin buffer

after gel is run, assemble transfer sandwich according to the following diagram

place sandwich into the transfer tank and transfer for 1 hr at 900 mA

the Towbin buffer should be cooled and stirred during transfer

remove sandwich and mark the position of the gel on the nitrocellulose

trim the nitrocellulose to a size somewhat larger than the gel and mark the position of the molecular weight standards

wash the western twice for 5 min in PBS-Tween and then dry on paper towels

the western can be stored for years between sheets of blotting paper

Evaluation of western blots

the efficiency of transfer can be evaluated by using pre-stained molecular weight standards or by staining the blot with Ponceau S

after transfer the blot should be washed with PBS-Tween as usual - Ponceau S is then briefly added to visualize transferred proteins

rinsing the blot with ddH20 leaves the proteins stained with Ponceau

to remove the Ponceau, wash the blot twice for 5 min with PBS-Tween

then dry the blot as usual on paper towels

you can also save the gel after transfer and stain it to determine the success of transfer - some proteins do not transfer well