Western blotting

SDS-PAGE/Western Blotting Protocol

Electrophoresis

<Preparation of SDS-PAGE samples>

Mix original samples and 2x SDS-PAGE Sample Buffer in the ratio of 1 to 1.

<Preparation of SDS-PAGE gel>

Separation gel

Mix in 50ml tube (15ml for 2 pieces)

Pour milli-Q water over the surface 

Stacking gel

Mix in 15ml tube (6ml for 2 pieces)

Fix for more than 1 hour

<1x SDS-PAGE running buffer>

Mix in 10l tank

Incubate the samples at 95C for 5min to denature.

Centrifuge the samples for 30 sec at 4C, 400×g to spin down the sample.

Set the gel in the electrophoresis apparatus and pour in SDS-PAGE running buffer.

Clean the wells by using a syringe. (If the wells are deformed, shape them by using loading tip.)

Load 5μl each of the samples and molecular weight marker into the wells

Run at 100V, 10mA per gel

After the samples and marker go past the stacking gel, change the setting to 100V, 100mA and run again. (This value is constant regardless of the number of gels.)

Electroblotting (Tank Transfer)

<1x Transfer buffer>

Transfer for 2h at 100V stirring continuously by a magnetic stirrer, in refrigerator. 

Blocking

<5% skim milk>

Pour 10ml of the skim milk in a plastic tray.

Pick up the membrane only with tweezers and immerse it in 5% skim milk.

Block the membrane in the skim milk for 1h at room temperature.

Primary antibody reaction

Discard the 5% skim milk and wash the membrane with PBS-T for 5 min.

Pour optimal amount of 5% skim milk in a plastic tray.

Immerse the membrane in the 5% skim milk..

Add primary antibody solution.

Incubate on shaker overnight at 4C.

Secondary antibody reaction

Discard the skim milk. Then, with PBS-T, rinse the membrane 3 times and wash it 3 times for 5 min.

After washing, discard the remaining PBS-T.　

Pour optimal amount of the 5% skim milk and add secondary antibody solution.

Incubate on shaker for at least 3h at room temperature.

Chemiluminescent detection

Discard the 5% skim milk. 

Then, rinse the membrane 4 times with PBS-T for 5 min.

Detect by appropriate chemiluminescent reagent (We use ECL-prime, Amersham).

<List of reagents used to prepare gels and buffers for experiments>

1. 5MTris-HCl(pH8.9) 500ml

0.5MTris-HCL(pH6.8) 500ml

30% AA sol

10% AA sol

2x SDS-PAGE sample buffer

10x Tris-Glycine, 1l

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