EZQuant–ELN Knowledge Base

Western Blotting	Table of Contents

Gel Preparation:

• Assemble the glass plates and spacers.
• Pour the running gel to about 1cm below the wells of the comb.
• Seal with ddH₂O.
• When the gel has set, pour out the ddH₂O.
• Pour the stacking gel and insert the comb immediately.
• Prior to running the gel, remove the comb and flush the wells out thoroughly with running buffer.

Running the gel:

• Mix the samples with SDS PAGE sample buffer and boil for 10 min.
• Load samples into the wells. Be sure to use a protein marker.
• Run with constant current (35-37mA). Some precast gels are run at 200V (constant voltage).

Membrane preparation:

• Cut a piece of PVDF or nitrocellulose membrane.
• When using PVDF membrane, wet the membrane for about 30 min in methanol on a rocker at room temp. Then, remove methanol and add 1x blotting buffer until ready to use.
• Nitrocellulose membranes should be immersed in 1x blotting buffer until ready to use.

Membrane transfer:

• Pre-wet the sponges and Whatman papers (slightly bigger than gel) in 1x blotting buffer.
• Assemble the transfer "sandwich": Sponge - Whatman paper - gel - membrane - Whatman paper – sponge.
• Transfer for 1 hr at 1A at 4°C on a stir plate. Bigger proteins might take longer to transfer.
• When finished, wash the membrane for a few seconds with Ponceau solution and rinse with dH₂O.
• Immerse membrane in a blocking buffer for at least 1 hr.

Antibodies and detection:

• Incubate with primary antibody diluted in blocking buffer overnight at 4°C or for 2 hr at room temperature.
• Wash 3 x 10 min with PBS+0.1% Tween 20.
• Incubate with the appropriate HRP-conjugated secondary antibody diluted in blocking buffer for 1 hr at room temperature.
• Wash 3 x 10 min with PBS+0.1% Tween 20.

Detect with ECL.