We tested for DNA damage by Western Blot against gH2AX (a marker for DSBs). Our positive control has 1 band, while the lanes where we expect DNA damage have 2 bands; what could be an explanation for this?
Hello Senne Van Hulle
One possible explanation could be γ-H2AX ubiquitination in response to DSB inducers. Following DNA double-strand breaks (DSBs), there could be ubiquitination, leading to the recruitment of repair proteins to sites of DNA damage.
The lack of attention to mono- (ub1) and diubiquitinated (ub2) products (γ-H2AX-ub1,2 ) forms probably reflects a wide use of γ-H2AX prior to the discovery of its regulatory ubiquitination and is further reinforced by the absence or near absence of higher bands on Western Blots provided by all major suppliers of γ-H2AX antibodies.
However, antibody vendors often use either UV (non-DSB damage at environmental doses) or the apoptosis inducer staurosporine for their positive samples. If γ-H2AX-ub1,2 are abundant but undetected products, then measurements of only γ-H2AX would give an incomplete assessment of the entire DSB response and may lead to inaccurate conclusions about DSB repair if γ-H2AX-ub1,2 and γ-H2AX display different decay kinetics.
You may want to read more on this subject by referring to the article which I have attached for your reference. (see Fig 1).
Article Monoubiquitinated γ-H2AX: Abundant product and specific biom...
So, probably the second band could be either γ-H2AX-ub1 or γ-H2AX-ub2 depending on the molecular size.
Best.
Malcolm Nobre, thank you for your answer. As you might have seen, if you search for this question on ResearchGate you find the same case mentioned 9 years ago. Remarkably no one suggested the answer you gave!
I should also add that our positive control was staurosporine for this experiment, which showed only one band as expected. Only 'bacteria-induced DNA damage' samples had double bands.
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