BioProtocols--WB for phospho protein

Western Blot of Phospho-Protein Protocol

Lyse the cells by scrubbing, or homogenize the tissue of interest in lysis buffer (e.g. RIPA) made fresh and containing a cocktail of protease inhibitors (and cocktail of phosphatase inhibitors when dealing with phosphorylated proteins). Sonicate the samples shortly, and then centrifuge for 20 minutes at 12000 rpm at 4C. Transfer the supernanant, and this is your sample for Western blot. Do protein concentration assay with the method you are familiar.

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 4C 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 inhibitors: Ready to use cocktails of inhibitors from well known suppliers are often used but you can make your own cocktail.

Remember to add phosphatase inhibitors to cocktails bought when investigating

phosphorylation events. Please remember that phosphatase is very active at your surrounding. This is why it is so difficult to detect phosphorylated-proteins.

1. To a sample of protein solution containing 1-100 ng of the target protein (500 ug lysate), add an equal volume of 2x SDS-PAGE sample buffer. For reduced samples, the sample buffer should be supplemented with DTT or ?-mercaptoethanol. For non-reduced samples, the DTT or beta-mercaptoethanol is not added.

SDS sample cell lysates can be stored at -20C for short-term use (less than 3 months), but should be kept at -80C for long-term storage. The degradation of activated protein samples varies greatly. It depends on the nature of each protein and how much activated protein is within the samples.

2. Denature the proteins by 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 or nitrocellulose membrane using semi-dry or wet transfer methods. PVDF membrane is highly suggested for WB on phosphorylated proteins. Please note: for PVDF it is essential to pre-wet the membrane in methanol prior to transfer.

5. If required, the efficiency of transfer can be determined by staining the membrane briefly 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.

6. Block the membrane with 5% w/v milk in TBST. Incubate for 1 h at 4C, with agitation.

7. Dilute the primary antibody in 5% BSA in TBST to the recommended dilution. We recommend incubating in a sealed bag, or hybridization tube. Incubate 1 hour at room temperature, or overnight at 4C with agitation. (We recommend to incubate the blot with primary antibody overnight at 4C).

8. Wash the blot in TBST with agitation three to four times for 5 min each at room temperature.

9. Dilute the horseradish peroxidase (HRP) labeled secondary antibody at the recommended dilution (1/3000--1/5000 is usually a good working dilution although this needs to be optimized for the particular application) in TBST.

10. Wash the blot in TBST with agitation three times for 5 minutes each, or four times with 5 minutes each at room temperature.

11. Perform ECL detection and develop the x-ray film.

Top tips for WB of phosphorylated proteins:

1. Keep the proteins in their phosphorylated state. Add adequate phosphatase

inhibitors and keep samples on ice at all times.

2. Block the membrane in 5% w/v BSA (fractionV) NOT MILK (milk contains casein which is a phosphoprotein; This is why it causes high background because the phospho-specific antibody detects the casein present in the milk).

3. Remember the phosphorylation may need to be induced. Low signal or no signal may mean that the induction is not sufficient. Run the recommended positive control with your samples.