Hi Everyone,
So I am trying to to do a Western blot from mouse spleen lysates of four different genotypes. With actin I get plenty of background whereas GAPDH works better. The problem is that in the two control genotypes I get a good amount of GAPDH but in the two experimetnal genotypes the GAPDH is greatly reduced. I am not sure whether this was an overestimation of the protein quantity or it is a genotype thing (I do have increased apoptosis). I run an SDS-PAGE and stained it by coomasie and to my understanding I load equal amount of protein. Can you suggest another housekeeping gene to look up. See the attached file for details pelase
β2-microglobulin is a good housekeeping control. However, loading amount needs to be optimized based on the method of detection.
Maybe, you can ignore housekeeping gene. See the link below.
https://www.researchgate.net/post/What_should_I_use_as_a_loading_control_when_western_blotting_Drosophila_hemolymph
Thank you all for your replies.
Jonas I cut the spleen in half, homogenize, measure the protein and try to load equal amount in the gel. The spleen has a heterogeneous population of cells but by doing this I try to be consistent between the mice.
The experimental spleens are larger due to the knocked down gene (which is expressed/not expressed in macrophages only) and therefore I cannot exclude that the difference in actin/gapdh could be due to the different genotype and not a technical issue.
So if I do Ponceau staining which band I should use for normalization and what if it gives a slightly different pattern similar to the coomasie gel? I wonder if I can use the lower bottom thick band of the coomasie gel for normalization
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