Western blotting quantification and normalisation - (Dec/08/2003 )
I would like to quantify signals from western blotting.
1. Should I repeat a sample (positive control) between all my gels?
2. What's the right way to normalise my data? Should I normalise in relationship with beta-actin (house-keeping) and also with my positive control?
Thanks for all inputs
You can quantify your western bands by gel densitometry. The software provided by Kodak is very good in this respect as it will also allow you to subtract the backgroundfrom the mean intensity. Good luck
after you've probed for your protein you need to strip the blot and then probe again with a housekeeping gene (we use alpha-tubulin, but b-actin should also be fine). once that's done you can scan the films and quantitate them with a densitometer. normally i divide the value the densitometer gives me for my protein band by the value for alpha-tubulin for that sample. so that you end up with a ratio of protein:tubulin for each sample and then graph that. it's an arbitraty value, but it allows you to compare between samples. i dont think you would want to normalilse your data to your positive control.