Rabbit muscle actin was purified from acetone powder (Pel-Freeze Biologicals) as described previously (Pardee and Spudich 1982 (link)). ADP-G-actin was prepared using hexokinase (Worthington Biochemical)-conjugated Sepharose beads as described previously (Makkonen et al. 2013 (link)). UNC-60A was expressed in Escherichia coli and purified as described previously (Ono and Benian 1998 (link)). DyLight 680-labeled actin was prepared as described for preparation of DyLight 549-labeled actin (Liu et al. 2010 (link)) using DyLight 680 NHS ester (Thermo Fisher Scientific). Histidine-tagged maltose-binding protein (MBP) was expressed from pDEST-HisMBP, which was developed by the group of David Waugh (National Cancer Institute, Frederick, MD) and obtained through Addgene, and purified as described (Nallamsetty and Waugh 2007 (link)).
Protocol detail
Purification of Rabbit Muscle Actin and Actin-Binding Proteins
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Acetone
Actin
Adp g actin
Biologicals
Escherichia coli
Ester
Freeze
Hexokinase
Histidine
Histidine binding protein
Maltose
Maltose binding protein
Muscle
Powder
Rabbit
Sepharose
Corresponding Organization : Emory University
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Variable analysis
independent variables
- Rabbit muscle actin purification from acetone powder
- ADP-G-actin preparation using hexokinase-conjugated Sepharose beads
- UNC-60A expression and purification in Escherichia coli
- DyLight 680-labeled actin preparation
- Not explicitly mentioned
- Not explicitly mentioned
- Positive control: Not mentioned
- Negative control: Not mentioned
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