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Phosphorylated Protein Detection Frequently Asked Questions
During the “Western Blotting Tips for Phosphorylated Protein Detection” Coffee Chat and “Illuminating the Pathway to Confident Western Blot Detection of Phosphorylated Proteins” webinar, researchers asked questions to Bio-Rad’s application scientists about the detection of phosphorylation events by western blotting.
To help you overcome this challenging technique, we have combined our answers for the most frequently asked questions.
Store your lysates at -80oC to prevent any degradation of phosphorylated proteins by phosphatases present in the lysate.
Use phosphatase and protease inhibitors and keep your samples on ice to inhibit protein degradation and conserve posttranslational modifications during sample preparation.
Start with bovine serum albumin (BSA) in tris-buffered saline (TBS). It is usually best to avoid using milk because some protein components may interfere with recognition of phosphorylated proteins. If using BSA, make sure it is thoroughly dissolved because any solid BSA will cause speckles on your blot.
A good starting point can be 5% BSA with 0.1% Tween 20 in TBS but this may need to be optimized for your protein of interest.
Consider the following controls:
Fig. 1. Western blot detection of total and phospho EGF R. MDA-MB-468 untreated and EGF treated whole cell lysates were probed with A, Human Anti-EGF R Antibody (VMA00474) or B, Human Anti-EGF R (pTyr1045) Antibody (VMA00515) followed by detection with HRP conjugated Human Anti-Bacterial Alkaline Phosphatase (HCA275P).
Try preparing your lysates in as little lysis buffer as possible. You could also immunoprecipitate your protein of interest before loading it onto your gel.
It is better practice to use the Stain-Free blot image for total protein normalization, since variability in transferring your proteins to the membrane could result in differences between samples not captured by the Stain-Free gel image. To reduce the background in these images, use low-fluorescent PVDF membrane.
The phosphatase you use depends on the type of phosphorylation event you are interested in. Both lambda protein phosphatase and calf intestinal alkaline phosphatase have an activity towards phospho-tyrosine residues. In addition, lambda protein phosphatase also has an activity towards serine and threonine phospho residues.
Use a conformation specific secondary reagent, like Bio-Rad’s TidyBlot Reagent, which only detects the native conformation of an antibody (i.e. your primary antibody) and not the denatured endogenous IgG in your samples. This means that the heavy and light chains from the antibodies in your sample will not be detected. The only band that should appear on your blot is from your antibody detecting the phosphorylated target.
We recommend performing a multiplex fluorescent western blotting experiment rather than stripping and reprobing blots. This is because the signal from the first phospho-specific antibody might not be completely removed during the stripping process. You can learn more about Bio-Rad’s fluorescent western blotting workflow here.