How can I load equal amounts of protein into each well during SDS-PAGE? - SDS-PAGE, WB, BCA assay, Actin (Jan/23/2008 )

BCA assay is used to make sure that I loaded the equal amounts of protein into each well. But I noticed that after western blotting, Actin still presents a decrease with lower concentration of total protein. And I don' t think our reagents influence the expression of Actin. But the reagents do inhibit the proliferation of cells, so we can only get the samples with decreased cell numbers (vary a lot).

Any advice to get the equal amounts of Actin as an internal control?

Thanks

-Twisters-

-Twisters-

Estimate protein conc. of samples using a kit and then calculate so that you load equal amounts.

-scolix-

you did a BCA assay to load equal amount of proteins?
Did you dilute your standard in the same buffer than your samples?

-Missele-

QUOTE (scolix @ Jan 24 2008, 06:38 AM)

Estimate protein conc. of samples using a kit and then calculate so that you load equal amounts.

Yes I did estimate protein conc. using BCA assay. But Actin band turned to be decreased in response to decreased cell number even with the same total protein conc. (Now I doubt if the results of BCA assay actually represent total protein conc.) This happened all the time.

Nobody encounters the same problem?

Thanks

-Twisters-

-Twisters-

QUOTE (Missele @ Jan 24 2008, 06:49 AM)

you did a BCA assay to load equal amount of proteins?
Did you dilute your standard in the same buffer than your samples?

Yes I did the BCA assay. But I use water to dilute BSA standards and the samples, are in 1X cell lysis buffer. Does that matter?

Thanks

-Twisters-

-Twisters-

You will have to dilute the BSA standards in the same buffer. How does your BSA curve look like? Is it linear? Did you recheck the calculations?

-scolix-

QUOTE (scolix @ Jan 24 2008, 01:31 PM)

You will have to dilute the BSA standards in the same buffer. How does your BSA curve look like? Is it linear? Did you recheck the calculations?

That sounds reasonable.

A recent BSA curve is: Conc.=10591*OD-735 (see attached curve)

The values will be calculated automatically.

Thanks

-Twisters-

-Twisters-

Hi one of my lab colleagues also encountered the problem of unequal beta-actin. she used water to dilute BSA as plotted her BSA graph. the R value is about 0.992. and although she used lysis buffer to lyse the cells and obtain the cell lysate which supposedly she uses as her sample for gel loading, she diluted the sample in 1:2, 1:5 and 1:10 dilutions (in water) in order to calculate her sample protein concentration. im not sure if i understood the posting so far, but in this case, the dilutions for both BSA and samples are done with water.or do u mean that she should have diluted everything in lysis buffer, even for BSA and the sample serial dilutions?

Pls advise. Was your actin more equal after using lysis buffer to dilute the BSA?

Thanks alot in advance..

-jae.tan-