Hello everyone! I regularly use the Sigma BrdU cell proliferation kit for my cell assays, and due to the nature of the kit I always run out of some reagents before others. This means I keep repurchasing the same kit just to replace one or two components since they don't sell them separately, and so I have accumulated multiple vials of the BrdU labeling solution as well as the wash buffer. It makes no sense to keep buying this kit since it is quite expensive. I would like to know if anyone has any experience in replacing the fix-denaturing solution, the anti-BrdU-peroxidase antibody, the antibody dilution solution, or the substrate solution, as a DIY reagent and still have the assay work just as well????
As a side note, I now use MTT instead of BrdU for cell viability because its much cheaper, however I am repeating a particular set of experiments from last year where I originally used the BrdU kit, so I would like to use the same method for consistency purposes here so I don't have to re-do everything with MTT....
Hi James:
I would encourage you to mix the components of the kits from different lots. To be cautious:
1) check the manual of the kits from different lots in case any specific changes
2) find the documents or original publications which explain the essential components in each reagents, most of them just normal buffer, but ridiculous expensive because of efforts on optimization and patent;
3) try to validate and optimize your new combinations under your lab conditions.
I have played with Click-iT Edu kit (invitrogen, C10337), chased down the original publication in PANS, found out the essential buffer recipe and made the buffers according it, diluted two main reagents 20x, it worked much better than original kit, and last for 4 years.
Hope this experience is helpful to you.
Regards
Shiqiu
yes, if its gonna be a standard assay of the lab I would even go so far as to just buy powder and make ur own buffer, those kits will have you manning a bakesale to keep costs. If not, like shiqui says, just mix it from the lots, if it has azide init and it's protected from light you can double the recc shelf life more than likely,.
One thing I tend to run out of quite quickly from that kit is the stop solution, and I've found that using either H2SO4 or HCl at standard ELISA concentrations works just fine, so that I can make the kit last a bit longer. Good luck!
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