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Western Blot: Actin bands too thick?

Western Blot: Actin bands too thick? - Western Blot Forum

Western Blot: Actin bands too thick? - 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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Old 03-05-2013, 06:17 PM
Pipette Filler
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Default Western Blot: Actin bands too thick?



New to western blotting, and trying to figure out the best way to do the loading control using beta actin. Because my protein of interest is not expressed at high levels, I am loading a lot of total protein in each lane to get a good signal. But that makes the actin bands really large. Is there a way around that?
Thanks!
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Old 06-16-2013, 04:16 PM
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Default Re: Western Blot: Actin bands too thick?

Quote:
Originally Posted by ajc613 View Post
New to western blotting, and trying to figure out the best way to do the loading control using beta actin. Because my protein of interest is not expressed at high levels, I am loading a lot of total protein in each lane to get a good signal. But that makes the actin bands really large. Is there a way around that?
Thanks!
Are your doing colorimetric or chemiluminescence? If its the latter then just expose for a shorter period to get smaller bands and hence see any differences. Either that or use your antibody at a lower concentration. Probably the secondary over the primary in my experience.
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