Western blot for phosphorylated proteins

Procedure for detection of phosphorylated proteins.

Procedure

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To a sample of protein solution containing 1-100 ng of the target protein (500 µg lysate), add an equal volume of 2x SDS-PAGE sample buffer.

For reduced samples, the sample buffer should be supplemented with DTT or ß-mercaptoethanol.
For non-reduced samples, the sample buffer should be supplemented with DTT or ß-mercaptoethanol. For non-reduced samples, the DTT or ß-mercaptoethanol is not added.

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Denature the proteins by heating the sample to 95°C, or boiling, for 5 min.

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Load the sample onto an SDS-polyacrylamide gel and run the gel under standard conditions.

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Transfer the proteins to a PVDF membrane using semi-dry or wet transfer methods.

For PVDF it is essential to pre-wet the membrane in methanol prior to transfer.

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If required, the efficiency of transfer can be determined by staining the membrane briefly (10 sec) in Ponceau stain.

The stain can be removed by washing in PBST or TBST.

We would recommend not washing blots in distilled water as this can strip off proteins in some circumstances.

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Block the membrane with 5% w/v BSA in TBST.

Incubate for 1 hr at 4°C with agitation.

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Dilute the primary antibody in TBST to the recommended dilution.

We recommend incubating in a sealed bag, hybridization tube or 50 ml Falcon tubes (~2.5 ml primary antibody/blot).
Incubate overnight at 4°C with agitation.

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Rinse the blot in TBST three to four times for 5 min each at room temperature.

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Dilute the horseradish peroxidase (HRP) labeled secondary antibody at the recommended dilution in TBST.

1/5000 is usually a good working dilution although this needs to be optimized for the particular application.

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Rinse the blot in TBST three to four times for 5 min each at room temperature.

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Perform ECL Plus detection.

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