4 replies to this topic

[dandan]

Hi folks

these days my western blot loading control is always acting up, never got equal band.
my protein quantification standard curve is perfect, but somehow, i dont know the actin is not equal.
we all know that western blot is not a real quantitative method, sometime my gut feeling is the more we
try to precisely quantify the protein concentration the worse result it comes out. some people even argue that there is no point to do protein quantification each time.
i have heard some lab protocols dont perform protein quantification, which helps the lab save a lot of money and time from this.what they do is try to seed the same number of cells when they set up the experiment and add same amount of loading buffer and load the same amount. but my concern is, is this really applicable to every cases? in my case, i need to treat drugs to kill or arrest the cell cycle, so different samples may have different numbers of cells by the end of the treatment,
therefore it may not a good idea to omit the protein quantification.
is there any better and steady way to get the loading control equal every time in western blot rather than this?

can anyone share some experience on tackling this issue.
thanks

dan

[mdfenko]

rather than get the loading control equal, you can use loading control ratios to normalize the samples.

Edited by mdfenko, 04 October 2011 - 12:39 PM.

[bob1]

You can also detach the cells, count, and resuspend in lysis buffer at a defined number of cells per ul.  This way, you know you are loading equivalent amounts of protein in each lane.

[dandan]

thank you guys for your replies and sharings.

[dandan]

bob1, on 04 October 2011 - 02:46 PM, said:

You can also detach the cells, count, and resuspend in lysis buffer at a defined number of cells per ul. This way, you know you are loading equivalent amounts of protein in each lane.

sounds good, will try it out, do people really practise like this? kekeke