quantification of WB bands- how to get similar values from different experiments - (Jun/21/2006 )
I need help with quantification of western blot protein bands.
I compare my protein of interest band intensity to a housekeeping gene. The problem is that this ratio varies significantly from one expreiment to another. I get a similar result when evaluated "by eye", so that it´s clear that one band is bigger than the other and the housekeeping gene bands comparable. But the final ratio is for example 0,1 in one experiment and 0,5 in another (which leads to a mean 0,3 with a standard deviation 0,3!!)
Any idea how to get so nicely consistent result (with nice error bars) with this method? I always admire the figures I find in papers! How do people do it?
Is this method worth anything anyway?
I guess you just have to keep increasing the n values. That's why normally the result is valid when the n values equals to minimum 3.
I´m not sure if it helps in my case. I already tried to do the same type of experiment some time ago and couldn´t make it work, despite n=6.
I just wonder what am I doing wrong to get so different ratios, even when it "looks" the same.
There are many variables to control in a western blot that may preclude getting a low standard deviation. Among them are exposure time, duration of antibody incubation and protein loading amount - even duration of electrophoresis and transfer may impact final results. I would consider just showing pictures of your gel, as thats how 99% of western blot results are depicted in papers. IMO numbers are great, however a western image has intuitive value to the reader that a bar chart may not.
I had this feeling about quantifying my bands- there are so many variables that don´t change linearily that it is really tough to get the same result each time.