Western Blot - Quantifying Tumor samples from two different blots - (May/18/2013 )
I'm new here. And I have a question regarding about quantifying bands on two separate membranes.
So I have a total of 13 protein lysate samples from tumors. Out of those 13, there are 7 control samples, and 6 treated samples.
We only have pre-casted 10 lane gels, so I loaded those 13 samples on two separate gels.
gel 1 has 3 control + 3 treated samples
gel 2 has 4 control + 3 treated samples.
I'd like to ultimately compare the treated samples to control.
After normalizing to the loading control (alpha tubulin), how can i properly combine the numbers from each blot so that I'd have 7 data points for the control group and 6 data points from the treated group? As of now, the trend between the blots between control and treated is consistent (blot 1 and 2 - ~40% decrease of treated compare to control,) but the raw value varies from blot 1 to blot 2, so significance was not reached when entered into graph pad prism.
I'm hesistant to rerun the samples on a gel with more than 13 lanes so i can run all the samples together because we have little of the lysates.
Any help would be appreciated!
You have hit upon the major failing in quantitation of western blots - it isn't comparable across blots, even if you keep all (and there are a lot of them) variables as similar as you can, you still tend to get variances between blots. The only way you can possibly compare is to normalize the samples to a loading control of some sort (e.g. B-actin) and then do stats on the normalized samples. However, to do this properly you will need several repeats of each sample, and you need to ensure that you choose an appropriate statistical test for low sample numbers.
You could add a standard priteinb(recombinant preferably) and load equal on both gels. After developing make the densities of the standard protein bands same on both gels and correct the test proteins accordingly.