Hello, actually the main application of 2-mercaptoethanol is for reducing proteins. So 2-mercaptoethanol can cleave the disulfide bonds that may form between thiol groups of cysteine residues. By breaking the S-S bonds, both the tertiary structure and the quaternary structure of some proteins can be disrupted. Also, 2-Mercaptoethanol can inhibit the oxidation of free sulfhydryl residues, and hence maintain protein activity. You can use try DTT but you need first to compare its chemical proprieties with the 2-mercaptoethanol and choose the one that fits your experiment the most.

Iskander Madhi thanks for your answer😁. But I still have some questions, hope you could help me. Do you think 2-mercaptoethanol or DTT can reduce the proteins that have already been oxidized? if so, does it mean those reductive agent could affect the primary antibodies bounding to my targeted oxidized protein?

I believe antibodies for WB are specifically designed to bind unfolded polypeptides in reducing conditions, that's why Abs used in WB are not capable of being utilized in other assays or haven't been tested by the ventor in other assays (if not mentioned). Sometimes though the recognisable epitope is not affected by the conditions, thus you can use these Abs elsewhere (I think mostly in histology, where proteins are denaturated by HIER). Maybe this topic might also help https://www.researchgate.net/post/What_is_the_difference_between_reducing_and_non_reducing_conditions

Dear Yayu

it depends from the properties of the primary antibodies that you are using. Are you using a policlonal and a monoclonal antibody?

In case of monoclonal, it depends for the kind of epitopes that are recognized. If the antibody recognize a conformational epitope which is lost when the protein is reduced because there is a folding change induced by the reduction therefore also the binding of the antibody is lost on the opposite if the antibody recognized a linear epitope that is preserved in case of reduction is it possible that the antibody do not feel any differences.

I think that the best way to discover it is load in the gel the reduced and non reduced samples and see if the WB provide same or different result for both samples.

good luck

Manuele

Filippos Theofilos Charitidis thanks a lot for your help. I’ll perform WB with 2-mercaptoethanol and IHC to see if the two results are at equal pace.

Manuele Martinelli thanks a lot for your answer. I’m using a polyclonal antibody, dose it mean the conformational epitope will have less influence on the recognition by antibodies?

Polyclonal just means that it was raised against a fragment of your protein of interest and is specific for any number of epitopes along that fragment. This approach to antibody generation is both cheaper and you are not dependent on a single epitope being present/properly folded but as a result is much more likely to bind nonspecifically to other proteins. In your case this means that your antibody is more likely to be able to bind the denatured protein than an untested/validated monoclonal antibody for your protein.

As Manuele Martinelli has said your best option is to run a gel and test it. Try to separate each reducing method by at least a few lanes since small molecules can diffuse through the gel across lanes!

If your only interest is identifying the protein on a western it might not matter if your protein is reduced or not so long as the reduced protein doesn't shift it's apparent molecular weight too much.

One final point I think is missing from this discussion is what exactly do you mean by an oxidized protein? Is it complexed with an ion that is the actually oxidized? Did you oxidatively stress cells and then collect a protein know to be oxidized? Did you isolate the protein and then oxidize the it outside of the cell? People could refer to each of these as oxidized proteins but each example has a different product.

I dont know

William Suslovic Thanks for your answer! I oxidatively stress cells previously then collect a protein know to be oxidized, and my purpose is to identify the oxidized protein.

I'll take your and Dr. Manuele Martinelli's advice. I very much appreciate your help.

Dear Yayu

as Wiliam told you policlonal antibody is a mix of antibody directed versus different epitopes and therefore in theory is it possible that it contains antibody directed versus the reduced protein as well as the oxidated and it is less subjected to it, howere it depends a lot from the nature of the immunogens used to generate the antibody because if it was used a peptite, a folded protein, a urea denatured protein becuase of course if the immunization was performed with a linear peptide there is less probability that it contain antibody able to recognize also the folded oxidatred protein respect than antibody produced immunizing the folded recombinant protein.

In generall antibody that are sold to use for FACS are able to recognize the folded protein while the antibody that are sold to use only in WB are able to recognize unfolded protein, but is it possible that there are exception.

good luck and happy new year!

Manuele