Thomas et al. (Anal Biochem 75, 177) recommended using the 3,3',5,5'-tetramethyl isomer, but googling for online recipes shows a number of labs are using N,N,N',N'-tetramethyl. Do they both actually work, and if so does one have any advantage over the other? Any information on interference by reducing agents DTT or 2-ME, or whether the samples should be denatured at room temperature or higher, (assuming covalently bound heme) would also be helpful.
I routinely use 2, 2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) as a gel heme stain, with sensitivity comparable (or better) than that afforded by N,N,N',N'-TMBZ.
I avoid the use of thiol reductants in the sample loading buffer, and samples are prepared by immersion in a boiling water bath for 2 minutes.
I can send further details of the staining protocol if required.
Nick Fisher
Thanks, Nick!
I'll get in touch by email to get the protocol.
We get good results with 3355TMBZ although we use 2ME, but (1) we never compared +/- 2ME, and (2) we denature at room temperature. I seem to remember that both both heat and reducing agents promotes dissociation of iron from heme, which would eliminate the peroxidase activity, so it may be that boiling with 2ME or DTT would be a problem.
Interestingly, in the same volume of anal. biochem. as the Thomas article, there is another paper describing detection of heme in gels by the red fluorescence of the porphyrin molecule. Fe quenches the fluorescence, so for this to work it is necessary to remove the iron. Sort of a complimentary approach, I guess. But even denaturing at room temp, when scanning gels for flavin fluorescence with the GE Typhoon, we routinely see weak-fluorescing bands that can best be explained as c-type cytochromes. Unfortunately the instrument does not have the exciting laser or emission filter that would be optimal for porphyrin.
Ed Berry
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