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Western Blot: only non-specific bands and not protein of interest

Western Blot Primary Antibody p53 Mouse

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#1 Epigeneticist

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Posted 21 March 2013 - 06:59 PM

Protein is from normal mouse epithelial cells in culture. I loaded 30ug protein on a mini gel and probed for p53 (FL-393 from santa cruz) 1:500 and all I get is non-specific bands. I do not see a band where my protein should be, 53kD. Will increasing (1:1000 ) or decreasing (1:100 or 300) dilution help? I have no problem detecting B actin or other proteins. Any recommendations on troubleshooting? Could it be low levels of p53? p53 degraded? Bad antibody, (does not make sense as it's a highly referenced antibody)?

Thanks

#2 bob1

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Posted 22 March 2013 - 12:25 AM

p53 is normally pretty low expression and very very quickly degraded (30 min half life). Try UV irradiation (100 mJ in a stratalinker will work, look for p53 within 8 hours) or drug induced for a + control.

#3 Epigeneticist

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Posted 22 March 2013 - 05:21 PM

I actually UV treated my cells yesterday and the western is incubating overnight. Hopefully I see p53 this time.

#4 Epigeneticist

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Posted 23 March 2013 - 06:49 AM

p53 induction by UV Treatment: After my cells were confluent I removed the media then kept lid off and placed the dish in the center of the hood. I closed the sash and turned on the germicidal UV light. I did this for 5s, 15s and 30s. After treatment I replaced media and harvested protein after 4 hours. I also had a control plate with no UV treatment. Control had no p53 expression, 5s UV had low p53 expression, and high p53 expression for the 15 and 30s treatments. I am glad it worked and now I know my antibody works too.





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