siRNA control by Western blot - how do I get equal loading concentration? (Sep/13/2005 )

Hello everybody!

I´m trying to verify my siRNA knockdown effects by using Western blot, but as seen with the (anti-) Actin control the protein loading concentration was never equal in the beginning.

Is there another way to mesure the protein concentration than doing it with NanoDrop, which I use? Even if I average over three times it obviously doesn´t work! (I add 2mikoliter each time)

Please help! I´m lost!

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hi
maybe you gonna find this answer quite silly, but you can do a bradford assay.
basic principle: with a reagent that bind peptidic bonds, you make a colorimetric measurement. coloration intensity depends on the number of bonds, directly linked to protein concentration.
you make a calibration with known concentratios of BSA, and analyze your samples after. i do a duplicate assay for each prot sample. And that's good.
I don't think i'm that understandable in this post... But if you need more info, don't hesitate...
Fred

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Hi Fred and thanks for the rapid answer. Bredford is of cause a good suggestion, I thoght about that. But somebody told me that you'd need a great amont (50 mikroliter) of proteins just for mesuring (instead of 2 mikro for Nanodrop). But if nothing helps I´ll go that way!

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hi
honnestly i make a dilution of 1/10 and use at most 2microliter of my sample. But this volume depends on the oriin of the proteins. I usually analyze protein from cells. what is your "source"?

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Hi!

My source are Cells, too. (HeLa) Friends of mine used the bradford - method but it didn´t work that accurately either!

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Hi;
I agree with Fred. A bradford would be good. You should use at least a double measurement of each sample.
I personally use 2 µl per sample and it´s enough. Maybe it depends on how and with which volume you lyse your cells. I worked with siRNA in HeLa too and used RIPA-buffer to lyse the cells.
If you load equal amounts of protein afterwards the actin control shouldn´t be a problem no more.

Cheers

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Hi,

Thanks for your help, I´ll try it that way tomorrow and than will see how it worked!

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hi
for a Well of a 6well plate, i resuspend cells in 80µl of lysis buffer and analyze 1 to 2µl...

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