I want to determine if a treatment alters heme synthesis in cultured fibroblasts. I have found several papers in which this was done using a QuantiChrome Heme assay kit, which I have. The papers report using a cell lysate and normalizing to total protein. The problem is that the buffer used for cell lysis is not specified and the kit just says to mix a sample with the reagent. Since a lot of heme is in the mitochondria, I'm worried that I might remove them if I do a detergent lysis and then a high speed spin to pellet insoluble material. I may try lysing in 1% NP40 and then doing a low speed spin just to remove large debris. If anyone has experience with this, I would really appreciate any suggestions. Thanks.
According to the publication in the attachment homogenizing your frozen sample in 1% Triton X-100 in TBS and subsequent centrifugation at 5000 x g to remove debris might do the trick.
Good luck with your experiment!
Regards, Christian
Article Heme Levels Are Increased in Human Failing Hearts
Hi Michael,
This is the protocol that I use:
https://openwetware.org/wiki/Smolke:Protocols/Heme_measurements
I have not tried in on mammalian cell cultures yet, but it works really well for yeast and other microbes.
Good luck!
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