When I do the experiment to determine the molecular weight of Bromelain by SDS-PAGE electrophoresis, my teacher ask me to add beta-mercaptoethanol into sample buffer. The role of beta-mercaptoethanol is to break all the disulfide bonds and denature the protein of interest. But I wonder, the target is to determine the MW of bromelain, so if we denature it and make it fractionated into subunits, then how can we determine the MW of it? Because when I look for the whole structure of bromelain, there is no such information like this. How can we make sure that the MW of bromelain we achieve from SDS-PAGE is of the whole structure or just a subunit of it (I cannot find any information about the structure or the subunit of bromelain).
Thank you!
I think when we add beta-mercaptoethanil into sample buffer with bromelain, and SDS also in the gel, they are responsible for dentaruing protein, cut the S-S bond, breakdown the space structure of protein and They don't cut the peptide bond of protein so your enzyme, bromelain is not cutted into small pieces and it will not effected to the MW of protein.
If the protein is not known to have disulfide bonds then adding the reducing agent is unnecessary. If it does it might be better to make it smaller, but this has to do with the separation itself. Look up for cysteine residues on the structure.
SDS-PAGE of proteins that have been reduced with mercaptoethanol is useful for measuring the monomer molecular weight. Reduction of the disulfide bonds is important for allowing the protein to become completely unfolded so that it migrates properly for its molecular weight.
If the protein is an oligomer, disulfide bonded or not, SDS-PAGE is not the best technique to use to learn the mass of the oligomer. The best methods to do this are SEC-MALS (size exclusion chromatography with multiangle light scattering detector) and analytical ultracentifugation. These are done with the native protein and do not require a reducing agent.
It is possible to use native PAGE to obtain the mass of the native protein, but the method requires the protein to be run on a set of gels with different acrylamide concentrations, and uses a set of markers of known molecular weight. The protein should not be reduced.
How much is the appropriate beta-ME final conc were added? (Reducing SDS-PAGE)
2X Laemmli sample buffer contains 5% by volume mercaptoethanol:
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006028F.pdf
bME should be replaced with DTT (Cleland's reagent) because the reducing potential of DTT is better suited to break protein S-S bonds. As additional advantage, it doesn't smell quite as bad ;-)
Movement of proteins through the gel is determined by charge, size and shape of the protein. The SDS binds to proteins at a uniform ratio, and thereby ensures a constant mass/charge ratio for all proteins. It also unfolds the proteins, ensuring uniform shape. Reducing agents help with the latter process. Therefore, separation is (largely) determined by the only remaining variable: size.
I agree that DTT is preferable to mercaptoethanol, but it is also much more expensive.
Yes it is more effective to use DTT than B mercaptoethanol
In absent of beta-ME, what should I add to Buffer RLT for RNA extraction from Human whole Blood ?
Dear Shuaibu you can use dithiotheretol DTT less toxic as alternative to beta ME
During lyaste preparation we forget add BME... We find some coagulate formation.. How can we rectify this issue...
@Adil Manzoor: Add the reducing agent and heat to 50 C. With some luck, the precipitate will dissolve.
Just a query: Does this BME causes any LCMS interference in intact mass analysis of a multimeric protein?
There is the potential to form a disulfide-bonded adduct with cysteine side chains.
2-Mercaptoethanol (BME) is a reducing agent and antioxidant that reduces the levels of oxygen radicals. It is usually added to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 5% concentration. This is done because BME cleaves the intermolecular disulfide bonds and denatures proteins.
§Protein samples are denatured by heating them with a detergent SDS and mercaptoethanol.
§The SDS binds strongly to the proteins and gives them a high negative charge whilst the mercaptoethanol frees sulfhydryl groups, thus yielding polypeptide chains carrying an excess negative charge and similar charge to mass ratio.
§This helps the resolution of proteins strictly based on their size during gel electrophoresis.
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