Protocol W.1

Western Blots

This protocol was developed for the BIORAD protein gel and transfer apparatus. The buffers can be used with any electrophoresis/transfer system.

 

Solutions

Transfer Buffer

25 mM Tris 3.03 g Tris base

192 mM glycine 14.4 g glycine

20% MeOH 200 ml methanol

up to 1 liter with Q

do not adjust the pH it should be 8.3

10X TBS

100 mM Tris 8.0 100 ml 1 M Tris pH 8.0

1.5 M NaCl 87 g NaCl

up to 1 liter with Q

to make TBST add Tween 20 to 0.1%

to make prehybridization solution add serum to TBST to 5%

Ponceau

mix approximately 0.5 g Ponceau-S in 500 ml 1% acetic acid

(Fisher# BP103-50)

Developing Solution

100 mM Tris 9.5 15 ml 1M Tris 9.5

100 mM NaCl 3 ml 5M NaCl

5 mM MgCl2 0.75 ml 1M MgCl2

up to 150ml with Q

To use, add 66 ml NBT and 50 ml BCIP to 15 ml of Developing Solution.

NBT (sigma N-6876) 75 mg/ml in DMF, and BCIP (sigma B-6777 (pToluidine Salt) 50 mg/ml in DMF. Store at -20°C.

Procedure

• Run a standard SDS-PAGE gel and soak the gel in Transfer Buffer--30 minutes for 0.75 mm gels and 1 hour for 1.5 mm gels. Soak the nitrocellulose along with the gel.

• Set up the transfer to nitrocellulose in 1X Transfer Buffer and transfer for 1 hour at 100 V or overnight at 30 V.

• Remove membrane and soak in Ponceau for 5 minutes. Destain in 1% acetic acid and mark the molecular weight markers. Do not allow membrane to dry out.

• Incubate membrane in TBST + 5% serum for several hours to overnight at room temperature. Alternately, the membrane can be incubated in PBS containing 5% Nonfat Dry Milk at 37° for 1 hour.

• Add the primary antibody in 10 ml TBST + 5% serum and incubate for several hours. If using Milk Block, add primary antibody in 1X PBS and perform all washes in 1X PBS.

• Wash 3X with TBST + 5% serum and add the secondary antibody in 10 ml TBST + 5% serum.

• Wash 3X with TBST.

• Add the developer solution and stop the reaction with 0.1 mM EDTA.