Isolation of functional lysosomes from skeletal muscle
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Abstract
Lysosomes within skeletal muscle function to degrade dysfunctional debris and initiate retrograde signaling pathways. We developed a method to isolate purified lysosomal fractions using small portion of skeletal muscle, eliminating the need for density gradients or lysosome-modifying agents, ensuring high lysosomal purity without compromising structure or function. By enabling functional analysis via acid phosphatase, cathepsin-B activity, and calcium release, this approach offers a powerful tool to study lysosomal roles in muscle physiology, disease, and exercise.
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Supplementary Fig. 1)
This data isn't linked anywhere.
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TRPML1 activity
This is a really nice result! Do you have a way to measure 'lysosome concentration' or do you just base everything off of normalized protein concentration? I'm wondering if there is a way to be quantitative about experiments like these from one batch to another.
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protein composition
It's really nice that this protocol is pretty agnostic to the source and doesn't require any modification of the natural biology!
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functional alterations.
How do you control for lysosomal quality here, or intactness from your isolation protocol?
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activity assay
For all of these assays, is the assumption that most of your lysosomes are intact? How do you then measure enzyme activity from the intact lysosomes? Do you assume that the substrates can all pass through the membranes?
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entrifuged at a higher speed to
Do you have details on what speed and for how long?
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This step was repeated two more times
This is a little unclear. Which step was repeated two more times? Just homogenization?
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