I often isolate myofibrils using a homogenization protocol, but it is very common to get bundles of myofibrils (specially from cardiac cells). I wonder why and how I could optimize this isolation so it yields higher amounts of single myofibrils. Maybe any enzyme treatment...
First I do need to know for which type of experiments are you trying to obtain these myofrils. Are these for functional experiments such as Force vs. pCa?
It is possible to isolate myofibrils mechanically using precision tweezers under a dissection microscope. First pin the whole muscle out in a petri dish, then fill the rest of the dish up with paraffin oil. The individual muscle cells can then be dissected out. I spent 18 months using this technique.
Hi Elsa,
Thank you. We perform this type of isolation in my lab, however, I'd like to optimize the myofibril isolation, which is below the cell.
Felipe, if I knew the composition of your solution, I would be able to help much more, but in general what sometimes is forgotten is that it is very important to not use KCL, instead use KMS, also replace ATP with CTP, this way you are not phosphorylating your contractile machinery. You should also have high EGTA, at least 20mM, and a very complete cocktail of inhibitors of proteases. Do the procedure with the solution being cold, with these measures, I think you will get way more myofrills and they will be way healthier!
Hi Marco. Thanks again. Normally my muscle samples are in 50% rigor + 50% glicerol + inhibitors. Then, I do the homogenization proceed in Rigor which has KCl but no ATP. EGTA in general is at 10mM, which considering that Rigor doesn't have Ca, makes its concentration virtually zero. I'll try to apply your suggestions, but what I often ask is what hold the myofibrils together? I wonder if some enzymatic treatment cold optimize their separation. Maybe you have an idea?
You will also improve tremendously the isolation by adding 0.1% or a bit less of Triton X-100, a non ioninc detergent that helps to "skin" and permebilizes the preparations. The high EGTA and Triton also help in the dissociation process. Just avoid light exposure during the incubation time with Triton of about 30-45 min and do it in cold. After this step, you can wash in a solution without Triton and pull it with tweezers and I think will be pleased.
Ok. We have triton here. We use it more with cardiac samples. I'll try your ideas and let you know later. Thanks for the discussion Marco!
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