Does anyone know how much tissue is needed of a mouse heart to obtain sufficient protein for western blotting? We would like to cut a mouse heart longitudinally and use one half for blots and the other for histology, but are worried that we will not obtain enough protein. Thank you.
The amount of protein in the heart is enough for your experiment. However, it is better to use similar amount for all samples for example 1 gram.
Also, the final amount of protein which you can extracted from the sample is dependent to the protein extraction method.
I would say 100 to 200 mg of heart tissue is enough. After centrifugation, you can get ~ 75 microliter of sample. From this you can use 5 to 10 microliter for Bradford method and the remaining sample will be for Wesern.
I am not sure about the quantity of heart tissue required for protein extraction, but i can say 25 microgram of protein is enough for a single blotting experiment. Good luck
Thank you all very much for the replies! I was worried we might have to switch to using a rat model, but it seems as if the mice will be ok. Thank you!
Usually what you need to do is to first estimate the amount of protein in the homogenate using Bradford method (homogenize the tissue in lysis buffer, prepare say 10 % homogenate) and then load about 20-22 mg/ml on the ID-SDS PAGE gel. This should be enough to be transfeered onto the blot.
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