Protein extraction from rabbit hearts

[Amiodaron]

Hi!
We are planning to apply proteomics to unveil cardiomyocyte protein expression patterns in distinct stages of heart failure. We want to use a rabbit model of heart failure. So does anyone know any protocols regarding the first steps of the proteomic-workflow in this specific experimental setting: retrograd perfusion of the heart, cardiomyocyte isolation, protein extraction...?

Thanks

[kiki06]

Wow! welcome to the forum. That is a pretty complicated procedure although certainly doable.

1) You would have to isolated the cardiomyocytes carefully.
This would entail looking up Pubmed to see what previous investigators did to extract viable cardiomyocytes.
Q: Do you really need cardiomyocytes carefully extracted? Or can you simply remove the heart and simply extract the proteins from whole tissue?


2) Protein extraction is quite easy once you got the cells. You could easily extact with a proteomics compatible lysis buffer. Dialyze the cells and get them ready in a buffer that allows 2-D separation. Hard part is going to be getting SAME amount of protein loaded for DISEASE heart failure cells and Normal cells.

3) Separate on 2-D. Get a fun robot to collect the spots. Send them all in for sequencing! and compare using scanning/sequencing CONTROL vs DISEASE!

hope that helps!
post here your response!

Kiki

[Amiodaron]

Hi Kiki,

thank you for your response. I have already scanned the available literature (there are just a few research groups dealing with that specific experimental setting), but the procedural approach here is quite different. It is indeed an important question, if the isolation of cardiomycytes is really necessary or if it would not be easier to prefer a whole tissue-extract. The problem of whole tissue-extracts thereby is the unavoidable contamination by other cardiac cells (fibroblasts, leukocytes, endothelial cells, vascular smooth muscle cells...), so altered protein expression patterns in cardiac tissue-extracts do not necessarily reflect an altered cardiomyocyte protein-expression status. Nevertheless, some scientist have chosen that way...

best regards