Isolation of muscle protein & depletion of acto-myosin components

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Isolation of muscle protein & depletion of acto-myosin components

Method Summary

This method describes how to isolate soluble muscle proteins (from heart or skeletal muscle), and prepare it for subsequent mass-spec analysis. Specifically, acto-myosin components are depleted from the sample to allow for mass-spec analysis of less abundant proteins.

Materials

Lysis buffer

  • 300mM KCl
  • 30mM PIPES pH6.6
  • 0.5% NP-40
  • 1x protease inhibitor (COMPLETE, EDTA free; Roche, Cat-No: 04693159001)
  • 1x phos-stop (Roche, Cat-No.: 04906837001)
  • 10mM Na-butyrate

Dilution buffer

  • 1x Phos-stop (Roche, Cat-No.: 04906837001)
  • 10mM Na-butyrate
  • 0.5% NP-40
  • 1mM DTT


Method

  1. Lyse hearts/muscle in ice-cold lysis buffer (approximately 500µl/30mg muscle; I used a polytron homogenizer)
  2. Centrifugate at max speed in a tabletop centrifuge (@ 4ºC) for 10 minutes & transfer supernatant into a new tube
  3. Dilute supernatant 1:4 with ice-cold dilution buffer (mix by inverting tube several times)
  4. Centrifugate at max speed in a tabletop centrifuge (@ 4ºC) for 15 minutes
  5. Transfer supernatant into a new tube & snap-freeze for mass-spec analysis.
  6. The pellet should contain the majority of the muscle acto-myosin and interacting proteins.

If everything worked out, a coomassie gel of the total samples (lane 1), the act-myosin myofibrillar components (lane 2) and the depleted supernatant mass-spec samples should look like the one here:

Coomassie of samples prepared by this method