Hi all,
I want to check the expression of phophorylated ZO1 and occludin from cell-lysate. However I couldn't get any Antibody commercially for phospho-ZO1 or Phospho-occludin as available for other junction proteins like phospho-VE-chadherine. Can anyone please suggest if any antibody commercially available for the same for western blot or any other method to achieve the same.
Thank you,
As you mentioned, there is no useful antibody which can detect phosphorylated ZO-1 and Occludin.
The slightly high molecular-weighted band which corresponds to phosphorylated protein should be detected as follows.
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.
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 DTT or ß-mercaptoethanol is not added.
Denature the proteins by heating the sample to 95°C, or boiling, for 5 min.
Load the sample onto an SDS-polyacrylamide gel and run the gel under standard conditions.
Transfer the proteins to a PVDF membrane using semi-dry or wet transfer methods. Please note: for PVDF it is essential to pre-wet the membrane in methanol prior to transfer.
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.
Block the membrane with 5% w/v BSA in TBST. Incubate for 1 hr at 4°C with agitation.
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.
Rinse the blot in TBST three to four times for 5 min each at room temperature.
Dilute the horseradish peroxidase (HRP) labeled secondary antibody at the recommended dilution (1/5000 is usually a good working dilution although this needs to be optimized for the particular application) in TBST.
Rinse the blot in TBST three to four times for 5 min each at room temperature.
Perform ECL Plus detection.
As said above, the phosphorylated form of your protein will migrate slightly slower on an SDS-PAGE gel as the non phoshorylated form. Your antibody (against the non phosphorylated form of your protein) will detect both bands corresponding to the non- and phosphorylated form of your protein. Pay attention to prevent dephosphorylation during cell extract preparation. You will have to choose the gel concentration to achieve the best resolution in the range of MW of your protein of interest. It may be wise to include a control with fully dephosphorylated cell lysate (CIP treated).
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