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Western blot for phosphorylated proteins

Procedure for detection of phosphorylated proteins.
Last edited Tue 31 Jan 2023

Homogenize the cells or tissue of interest in lysis buffer made fresh and containing a cocktail of protease inhibitors (and phosphatase inhibitors when dealing with phosphorylated proteins).

As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down tremendously if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer.

Use a RIPA or NP40 buffer supplemented with fresh protease and phosphatase inhibitor cocktails.

Remember to add phosphatase inhibitors to cocktails bought when investigating phosphorylation events.

Stage 1 - Procedure

Steps

1

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.
2

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

3

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

4

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

5

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. 
6

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

  •  Incubate for 1 hr at 4°C with agitation.
7

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.
8

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

9

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

10

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

11

Perform ECL Plus detection.