I did the subcellular fractionation of transfected HEK-293T cells.
For western blot analysis for the purity of fractions, I used anti-B actin antibody, anti-fibrillarin G4 antibody and anti-calnexin antibody for cytoplasmic, nuclear and membrane fractions, respectively.
Anti-B actin gave expression both in cytoplasmic and nuclear fraction. Later, I realized that B actin is also present in the nucleus!
With Anti-Fibrillarin, expression was detected in nuclear fraction only.
However, with Anti-Calnexin antibody, expression was both in nuclear and membrane fraction, with higher amounts in the former.
I was wondering, if that could be possibly be due to the contamination of nuclear fraction with membrane fraction.
Could anyone suggest me more specific markers for identifying the three fractions?
Thanks in advance.
Hello Smriti Chand
1. Yes, you are right. B-actin is also present in the nucleus, as a component of chromatin remodeling complexes. You may use α-tubulin as a marker for the cytoplasmic fraction.
The soluble α/β-tubulin is considered to be a reservoir of subunits that are available for microtubule polymerization in the cytoplasm, but there are reports supporting the presence of tubulin in the nucleus of cultured animal cells. The reason for nuclear accumulation of tubulin, which does not occur under normal circumstances, is a process of pathophysiological significance and may represent a defense mechanism against stress or malignant transformation. This is the reason why nuclear tubulin may be detected only in cancer or in transformed cells.
2. Calnexin is highly abundant in ER as well as the outer nuclear membrane. It has also been found at ER-mitochondria contact sites. But the possibility of contamination of nuclear fraction with membrane fraction cannot be ruled out. Nevertheless, you may try caveolin-1 for the membrane fraction. Caveolin-1 is a plasma membrane protein that has a fundamental role in the formation of caveolae and acts as a connective protein that organizes the macromolecular complexes on the cell surface which participates in intracellular signaling.
3. In addition to fibrillarin, you could use histone (H3) for the nuclear fraction. H3, one of the core histones along with H2A, H2B and H4 is known to form nucleosomes with nuclear DNA. It plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis.
You may try these markers.
Hope it helps!
Best.
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