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!
I have been doing similar experiments with macrophages. My question is if it is OK to get cell lysate before mixing it with Oxalic acid by using RIPA buffer with protease inhibitor cocktail. This buffer is usually used for Western Blot. I have the concern that we may loose some heme-containing proteins without using protease inhibitors.
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