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A micro-volume adaptation of a stopped-flow system; use with μg quantities of muscle proteins

Walklate, Jonathan, Ujfalusi, Zoltán, Behrens, Vincent, King, Edward J., Geeves, Michael A. (2019) A micro-volume adaptation of a stopped-flow system; use with μg quantities of muscle proteins. Analytical Biochemistry, . ISSN 0003-2697. (doi:10.1016/j.ab.2019.06.009) (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided)

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Abstract

Stopped-flow spectroscopy is a powerful method for measuring very fast biological and chemical

reactions. The technique however is often limited by the volumes of reactants needed to load the

system. Here we present a simple adaptation of commercial stopped-flow system that reduces the

volume needed by a factor of 4 to ≈120 μL. After evaluation the volume requirements of the system

we show that many standard myosin based assays can be performed using <100 μg of myosin. This

adaptation both reduces the volume and therefore mass of protein required and also produces data

of similar quality to that produced using the standard set up. The 100 μg of myosin required for

these assays is less than that which can be isolated from 100 mg of muscle tissue. With this reduced

quantity of myosin, assays using biopsy samples become possible. This will allow assays to be used

to assist diagnoses, to examine the effects of post translational modifications on muscle proteins

and to test potential therapeutic drugs using patient derived samples.

Item Type: Article
DOI/Identification number: 10.1016/j.ab.2019.06.009
Uncontrolled keywords: rapid mixing, myosin, actin, troponin, myofibrils, Ca regulation, muscle biopsies
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Sue Davies
Date Deposited: 21 Jun 2019 07:31 UTC
Last Modified: 01 Jul 2019 09:24 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/74516 (The current URI for this page, for reference purposes)
Geeves, Michael A.: https://orcid.org/0000-0002-9364-8898
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