I am measuring the level of a protein in different genetic backgrounds.
For example, I am measuring the level of protein X in wild type and atg8mutant.
I ran both the wild type and mutant samples on the same gel. Probed initially for protein X, followed by actin (loading control).
Bands were analyzed using an analysis software (Total lab).
Then I did a couple of normalizations.
1) Level of protein X was normalized to respective action control.
2) The control (wild type) condition was normalized to 1 and all other experimental conditions were compared to this.
Following is an example of what I have done.
SDS fraction Vol of protein x Vol of action
Wild type 695432.72 174080.04
Atg8a mutant 948245.24 61598.79
1) Normalisation- Vol of protein x in lane A divides by Vol of actin in lane A
That gives wild type =3.99
atg8a mut=15. 39
2) Relative protein level in relation to wild type -(Divide each sample with control (wild type) including the control)
That gives wild type =1
I repeated the experiment two more times and analyzed the data as described above. Other two experiments also follow the same trend (mutant with more protein). Since these data are from three independent experiments (three different western), How should I apply the statistics?
Should I use the values normalized with actin ( above described first value-3.99 and 15.39) or the value normalized to the 1 (1 and 3.85) for analysis?
Should I do a paired t test? Or two way anova (each experiment as random variable and group (wild type and mutant) as fixed variable?
Thank you very much. Any help will be appreciated.