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Western Blot Forum Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


Ubiquitinated Protein

Ubiquitinated Protein - Western Blot Forum

Ubiquitinated Protein - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 07-11-2008, 12:52 PM
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Default Ubiquitinated Protein



Hi,

Has anyone tried to use an antibody to detect ubiquitin or ubiquitinated protein on a western blot? If you see bands above your protein of interest on a western blot (very close to your band of interest and many of them) could it be ubiquitinated protein targeted for degredation or could it be something else?
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Old 07-11-2008, 03:33 PM
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Default Re: Ubiquitinated Protein

Yes I use anti-ubiquitin from Abcam, and it binds to many ubiquitinated proteins, resulting in many bands or an extensive smear on western blots.
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Old 07-11-2008, 07:12 PM
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Default Re: Ubiquitinated Protein

You can confirm that your specific protein in ubiquitinated by doing an Immunoprecipitation with one antibody (ie protein specific) and then the Western with the anti ubiquin (You also want to confirm by doing a Western with the protein specific antibody).
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Old 07-13-2008, 03:25 AM
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Default Re: Ubiquitinated Protein

How much molecular mass does ubiquitinylation add to a protein. If you section the membrane at the standards in the range of the protein of interest can you determine if it is ubiquintinylated by its location on the membrane.
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