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Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: disputes123 on October 16, 2019, 04:18:17 PM

Title: western blot for phosphoproteins?
Post by: disputes123 on October 16, 2019, 04:18:17 PM

I'm running a western on phosphorylation of a protein, and I'm wondering how do people get westerns with phosphorylated protein and total protein (non-phosphorylated and phosphorylated of that same protein) to act as a loading control rather than actin. All the papers I'm reading just says they incubated the membeane with the anti-phospho antibody and also the anti-protein antibody.

Not at the same time right? Because phosphorylated protein and protein itself is so little difference in mass how can you differentiate the bands?

Are people stripping the membrane then treating wity a different antibody?

Title: Re: western blot for phosphoproteins?
Post by: Yggdrasil on October 16, 2019, 04:53:20 PM

Yes, typically we will stain for the phospho-protein, strip the membrane then re-stain for the total protein.

Title: Re: western blot for phosphoproteins?
Post by: Babcock_Hall on October 16, 2019, 05:25:18 PM

I am completely unfamiliar with Western blotting; therefore, this information may or may not be useful.  However, I have seen at least one gel in which the phosphoprotein migrated differently from the non-phosphorylated form in an electrophoresis experiment.  IIRC it was Spo0F, and one of the coauthors was Jim Hoch.  I am not sure why the mobilities were different.