Dear ResearchGate community,
I hope this message finds you well.
I am currently investigating acid uric membrane transporters and their potential dimerization, both as homo- and heterodimers. One of the challenges I face in this study is that the monomers exhibit very close molecular weights, which complicates the identification of dimers in Western blot analyses.
In my previous experiments, I observed bands that may correspond to monomers, dimers, and what appear to be non-mature proteins. Additionally, some of the bands suggesting dimer formation are shared between different transporters, further complicating the interpretation of the results. Given the close molecular weights of these proteins, I find myself lacking sufficient evidence to definitively determine whether the bands correspond to homo- or heterodimers.
I would greatly appreciate any insights or recommendations on how to improve the detection and characterization of these dimers in my Western blot experiments. Specifically, I am interested in strategies that might help differentiate between homo- and heterodimers despite their similar molecular weights. Any tips on optimizing conditions or alternative methods to validate dimer formation would be invaluable.
Thank you for your time and assistance. I look forward to your guidance.
Best regards,
Ignacio G Barroso
Department of Tropical Medicine and Infectious Disease Tulane University
To separate intact oligomers, you need to use native electrophoresis. You can then separate the components by a second, denaturing electrophoresis. Such 2D approaches have been described, for example, in DOI:10.1006/abio.1994.1112. You can improve the resolution somewhat by using gradient gels, which narrow the bands by counteracting diffusion.
If you have antibodies against the different monomers, analysis of the blots is simplified. You could even use “rainbow” westerns to stain them in different colours on the same blot (DOI:10.1006/abio.1996.0160)
Thank you for your response.
I ran gels without Beta-ME, and one of the transporters displayed a band with double the expected molecular weight, suggesting it might form a dimer stabilized by disulfide bridges. However, the other two transporters did not show any bands. In the absence of Beta-ME, no bands were observed, while three bands appeared when it was present.
I am also considering using 2D electrophoresis to differentiate between the transporters, as their molecular weights are quite similar. I appreciate the reference!
We have gradient gels (4-15%) that I can use for testing; I had been using 10% based on the transporters' molecular weights.
I do have antibodies for each transporter, and I’ll check out the reference you provided to learn how to perform a rainbow Western blot, as I’ve never done one before. Thank you again!
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