WB Shows 20kDa Increase, How to Use Dephosphorylation and Deglycosylation to Confirm Post-Translational Modifications
In Western blot (WB) experiments, an observed increase of approximately 20 kDa in the molecular weight of a target protein compared to its predicted mass may suggest the occurrence of post-translational modifications. To verify this possibility, the following steps can be taken:
Dephosphorylation Verification
1. Sample Preparation
Ensure that a sufficient amount of protein sample is available for analysis.
2. Selection of an Appropriate Phosphatase
Alkaline phosphatases, such as Lambda protein phosphatase, or other enzymes specifically targeting phosphorylated residues are commonly employed.
3. Phosphatase Treatment
Follow the manufacturer’s protocol to incubate the protein sample with the selected phosphatase in the recommended buffer system for an appropriate duration.
4. Repeat Western Blot Analysis
Analyze the treated sample again using WB. A reduction in the apparent molecular weight, approaching the theoretical size of the protein, would suggest that phosphorylation may be responsible for the observed shift.
Deglycosylation Verification
1. Sample Preparation
Similarly, ensure the availability of adequate protein sample for enzymatic treatment.
2. Selection of a Deglycosylation Enzyme
Commonly used deglycosylation enzymes include PNGase F and O-glycosidase, which are capable of removing N-linked and O-linked glycan chains, respectively.
3. Deglycosylation Treatment
Perform the enzymatic digestion according to the supplier’s instructions, typically by incubating the enzyme with the protein sample in a specified buffer system.
4. Repeat Western Blot Analysis
Subject the treated sample to WB analysis. A decrease in molecular weight, aligning with the theoretical mass of the unmodified protein, may indicate glycosylation as the underlying modification.
When conducting these experiments, it is essential to include both untreated (control) and enzyme-treated samples for direct comparison, as well as a negative control group to validate the specificity of the observed changes. Moreover, optimizing reaction conditions—such as buffer composition, incubation time, and enzyme concentration—is crucial for achieving reliable and reproducible results.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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