Can anybody suggest the optimal concentration and time needed for MG132 treatment, in Hela cells transfected with protein that are presumably ubiquitinilated?
Hi,
in some cell lines I noticed that even 5uM for 4 hours is enough. Just make a little calibration with different concentration of MG132 and use poly-Ub K48 antibody in western blot in order to confirm the inhibition of proteasome
Greetings to all, I just noticed this thread and I was wondering what confluency is the best to start the MG132 treatment? any thoughts?
Hey guys!
I have a VERY long lived protein. I think that phosphorylation is regulating it's stability. I am trying to figure what pathway regulates the protein of interest's demise. Therefore, can you really treat cells with MG-132 for 24 hours?? Thanks!!!
Hi, can I use MG132 on fission yeast? If yes, how long I need to treat the cells?
Hi all, I transfected 2 types of cells (breast cancer cells and Hek293: both cells have good levels of the transfected constructs rna, but I can only detect protein in hek cells and not the cancer cells. So my question, is if I use the MG132, How long should I treat them before harvesting for protein to be seen by Western? thanks
we are using 50uM, 4 hours for HCT116 cells. very long hours of MG132 treatment can cause apoptosis (accumulation of p53 has this effect)
Can anyone suggest me what conc of MG132 should I use for HepG2 cell line?
Hello everyone, I am working on a project for hiv 1 integrase. I have performed cloning and according my seq results it was successful. It has been few weeks I’m trying to detect my protein on western but it doesn’t show up. Hiv 1 integrase is getting degraded so I am using proteasome inhibitor. I have tried several treatments but none of them has worked. I did 50ul for 3 and 4 hours, 20ul for 4 hours, 10ul for 8hours and 6hours, 2ul for 18hours. I seed the cells and thennafter 24 hours I make the transfection with my pcmv5 flag integrase plasmid and then after 24 hours I start the treatments. Cell line used is Hek cellsZ any advice is valuable. Thank you
I like to know how do you lyse cells for WB in order to make lysates since some protein get degraded easily with harsh method.
Hi, I want to ask please about the time of using, as I understood you need to optimise the dose of the MG 123 before using. My question is do you treat the cells after or before transfection? If after is it immediately? My problem is I detect the protein of interest in the cell lines not in primary cells.
Generally, we are using 40 µM in HEK293T for four hours. Some times we also use 30 µM for 8 hrs in HEK293T to do ubiquitination assay.
Hi Panagiota, I think your problem may be the cloning. Since HIV mRNA will contain splicing sites. In the presence of rev protein, it can be transferred in to cytosol for translation. But since you are expressing integrase separately, its mRNA may be considered as immature mRNA and subjected to splicing. I think that's why you can't get your protein detected with WB.
@Esra, it is done after the transfection, usualy 2-8 hours prior to collect the sample.
Hello everyone. I saw this thread and i thought i might get some advise here. I would like to use MG-132 to inhibit the proteosome in isolated cardiomyocytes. What is the optimal protocol (concentration and time)? Will highly appreciate. Thanks
@ Ruth: I would recommend trying different doses like 10-100 uM (final concentration). Usually I add MG132 in the cell culture media (could be with serum) prior to 2-8 hours of sample collection depending on my target protein, ideally 4 hours was OK for all. In summary, I added 20uM (Final concentration) DMEM media for HEK293 or MEF, incubated for 4 hours at the incubator and collected the samples for WB. Hope these helps. By the way, I was at UniPD (Padova) during 2015 working with Professor Dr. Luca Scorrano. It is a nice city.
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