I am going to do western blot for the nuclear fraction of mouse embryonic stem cells (E14 cell line).
What are the nuclear loading controls that are commonly used?
I know that Lamin B1 is a common nuclear control, but I have seen in some papers that Lamin A/C have some roles in ESC
http://www.ncbi.nlm.nih.gov/pubmed/23324457
http://www.ncbi.nlm.nih.gov/pubmed/16179429
I am not familiar with the works of Lamin, so I don't know how Lamin B1 is different from Lamin A/C. Would it be a problem if I use Lamin B1 as the control? Has anyone used it in ESC?
Hi Kc,
I don't have experience with Lamins as a nuclear loading control, however, according to this paper:
http://www.ncbi.nlm.nih.gov/pubmed/22116031/
Lamin B1 is not a good idea for ESC. As you already found out, Lamin A/C is expressed in ESC and seems to be connected to pluripotency, so it probably works. However, as a loading control for nuclear proteins I always use a protein that is (almost) expressed in any cell like the histones e.g. histone H3 (pan = recognizes all H3 regardless different modifications).
Good luck with your experiments
Ralf
Similar to whole cell fractions, loading controls can be a little iffy (e.g. using GAPDH). In my lab, I encourage the use of multiple controls. So for your situation I wouldn't use laminin as it is expressed on the nuclear envelope and there is much variance in its expression. Some other ones that you can use are Tata binding protein (TBP), histone H1, or PCNA. I would go with TBP and PCNA personally though.
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