Myosin S1 Fragment (Skeletal), Rabbit skeletal

On Arrival: 4°C
Briefly centrifuge to collect the product at the bottom of the tube. Reconstituting a tube of MYS04 with 75 ul of Milli-Q water will generate a 3.3 mg/ml stock of psoas S1 myosin in the following buffer: 20 mM PIPES pH 7.0, 1 mM EDTA, 5% (w/v) sucrose and 1% (w/v) dextran. The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4C desiccated(< 10% humidity) for 1 year.

* Limited stock available. If stock is not available, Cytoskeleton will produce a new batch upon request. Minimum order will apply. Inquire for more information.

Measurement of F-actin activated myosin ATPase activity
Identification/characterization of proteins or small molecules that affect myosin ATPase activity
Identification/characterization of proteins or small molecules that affect myosin / F- actin interaction

The biological activity of rabbit psoas myosin S1 fragment can be determined from its rate of F-actin activated ATP hydrolysis. The assay is constructed by first polymerizing actin to form F-actin, then the cardiac tropomyosin/troponin complex (TT complex) is mixed with the actin filaments in a stoichiometric amount. This creates coated filaments which are analogous to the thin filaments of muscle fibers. Myosin is added in substoichiometric amounts and the reaction initiated with ATP and calcium. Stringent quality control ensures that in the absence of calcium the TT complex completely inhibits myosin ATPase. Upon addition of 2 mM calcium, myosin ATPase will be restored (see Figure 3). Calcium binds to Troponin C which dissociates from F-actin allowing myosin to bind.

The following major steps are recognized:
Step 1. Assemble required reagents and compounds. (30min). Only if screening.
Step 2. Prepare F-actin polymer stock. (2h).
Step 3. Prepare Motor Mix and plate reader. (15min).
Step 4. Pipette Motor Mix into wells and start reaction/plate reader. (10min).
F-actin polymer stock
1. Resuspend ADMK or AD99 with 2.5 ml of Buffer to 0.4 mg/ml, Buffer is 5mM Pipes-KOH, 100 uM ATP, 500uM DTT.
2. Place at RT for 30 min to depolymerize the actin oligos that form during concentration/lyophilization.
3. Then add 2 mM MgCl₂and 2 mM EGTA and incubate at RT for 1 h to polymerize. (shelf life 1h at RT).
Myosin ATPase assay
1. Dilute S1 myosin to 1 mg/ml with ice cold PM12 Buffer.
2. Resuspend TT05 on ice with ice cold water to 5mg/ml. (100 ?l per vial for 0.5 mg vial).
3. Mix the following to make 1 ml of actin/TT/myosin (ATM) control mixture:
150 ?l of F-actin (0.4 mg/ml in 5mM Pipes pH 7.5, 2 mM MgCl2, 58 ?M CaCl2, 2 mM EGTA, 100 ?M ATP, 500 ?M DTT)
100 ?l of TT05
80 ?l of PM12 Buffer
100 ?l 5x MSEG
5 ?l of 100x PNP
4. Wait for 10 min to make thin filaments.
5. Add 2.5 ?l of S1 myosin and 5 ul of 50 mM ATP and mix.
6. Incubate at 37C for 3 min.
7. Using the pre-warmed half area 96-well plate, pipette the following:
8. Pipette 10 ?l of 2 mM calcium into "activated" wells.
9. Pipette 10 ?l of Milli-Q water into "non-activated" wells.
11. Pipette 90 ?l of myosin/TT/actin mixture into all wells.
12. Start protocol, 41 readings, 30 seconds apart, 37C , OD 360nm.
13. Calculate Vmax and compare non-activated to calcium activated samples.

Diagrammatic representation of the myosin protein and its subfragments Legend: Myosin is a hexameric protein consisting of two heavy chains and two light chains. Myosin can be proteolytically cleaved into heavy meromyosin (HMM) and light meromyosin (LMM) by ?-chymotrypsin in the presence of magnesium. In the presence of EDTA, however, ?-chymotrypsin produces the soluble myosin S1 fragment (3).