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B-actin as western blot -control


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#1 Kriz

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Posted 11 July 2010 - 05:37 PM

Hi! I am doing western blot to find the protein expression of my genes in normal and cancer tissues but I find that b-actin expression very less in normal and high in tumor samples. Usually since it is a house keeping gene, its expression level should be same in both normal and cancer tissue. Please help.

#2 WYF

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Posted 13 July 2010 - 01:30 AM

Although we tend to think the so called "house-keeping" genes should not change with conditions, they do. Perhaps b-actin does change in your tumor samples, if you are sure that you have loaded equal amount of protein...I would try it again, just in case i have done my maths wrong, or loaded more/less volume of the samples, or some how i haven't got an equal transfer...and if the problem persists, you might want to explore other house-keeping genes as loading control...

#3 deep

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Posted 15 July 2010 - 04:41 PM

HI kriz,
I think there unequal loading of proteins thats why beta actin not exact in each sample.
may be mistake in calculating protein conc . or in protein assay. check out this two possibilities..

#4 Kriz

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Posted 19 July 2010 - 09:21 AM

Although we tend to think the so called "house-keeping" genes should not change with conditions, they do. Perhaps b-actin does change in your tumor samples, if you are sure that you have loaded equal amount of protein...I would try it again, just in case i have done my maths wrong, or loaded more/less volume of the samples, or some how i haven't got an equal transfer...and if the problem persists, you might want to explore other house-keeping genes as loading control...



Yes equal amounts of protein was loaded, however where u require 7 microlitres for tumor, normal sample requires about 30 microlitres. does it effect when the wells are loaded almost full to its capacity? Will it affect the resolution and weaker bands?

#5 mdfenko

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Posted 19 July 2010 - 09:48 AM

ideally, you should adjust the volume of all your samples so that you load the same volume per well.
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