MIME-Version: 1.0 Content-Location: file:///C:/21C9598E/Fluorescentlabelingofsmoothmusclemyosin.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Fluorescent labeling of smooth muscle myosin2:

Fluoresc= ent labeling of smooth muscle myosin2:

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Myosin is selectively labeled on thiol groups present = in cysteine residues by coupling with fluorescent deriva= tive of maleimide.

 

1.&n= bsp;     1-2 ml of purified myosin at 5-10 milligrams ml-1

= 1.      10 mM DTT is added to either frozen purified myosi= n or to myosin ammonium sulfate pellet dissolved in Labeling Buffer (50 mM HEPES, pH 7.6, 0.5 M KCl, 1 mM EDTA),  and dialyzed overnight against  50= 0 ml of this buffer at 4o.  Dithiothreitol produces complete reduction of thiol groups, but must be dialyzed away to prevent interference with the subsequent reaction.

= 2.      2. Centrifuge the dialyzed myosin at 50,000 rpm in a Beckman TLA-100.3 rotor (k=3D64) for 15 minutes at 4o to clarify.

= 3.      Measure the concentration of myosin by the absorbance at 280 nm of a 1/20 dilution.  Calculate the milli= grams of myosin.

= 4.      Add a five-fold molar excess of tetramethyl rhodamine maleimide (TMR-= MI, Molecular Probes, Eugene, OR) from a 5 mM (2.4 milligrams ml-1) stock in dry DMSO with rapid mixing. The s= tock is stable for years frozen at –80o.  The ratio of the molecular weight = of TMR-MI to that of myosin =3D 482/5000,000=3D 1/1= 037.  Thus for a five-fold molar excess = of TMR-MI, you need 1/207 of the weight of myosin, e.g. for 25 mg of myosin, a= dd 0.12 mg of TMR-MI or 50 microliters of the 2.4 = mg/ml stock.

= 5.      Incubate for 1 hour at 4o.

= 6.      Add 1 mM dithiothreitol= to stop the reaction.

= 7.      Apply to a 5 ml Excellulose column (Pierce, Rockford IL) equilibrated with Column Buffer  (5 mM<= /span> PIPES, pH 7.0, 0.45 M KCl). Collect 0.5 ml frac= tions. Free dye will be retained and TMR-myosin excluded. The peak TMR-myosin frac= tion is apparent by maximum color intensity and should contain greater than 50 %= of the original myosin concentration. The dye-to-protein ratio is typically 3-4 calculated using an extinction coefficient of 50,000 M-1cm-= 1 at 558 nM for conjugated TMR and an extinction coefficient of 280,000 M-1cm-1 at 280 nM for myosin. The correction for the contribution of TMR to absorbance at 280= nm is 0.3 times the absorbance of TMR at 558 nm.

= 8.      Store TMR-myosin by drop freezing aliquots in liquid nitrogen

 

Solutions: Store at 4o.

Labeling Buffer: 500 ml=3D Na HEPES 6.51 g, KCl 18.64 g, Na2EDTA 0.17 g; titrate to pH 7.6.

Column Buffer: 100 ml=3D PIPES 0.15 g, KCl= 3.35 g; titrate to pH 7.0.

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2 This proto= col is a modification of A.B. Verkhovsky and G.G. Borisy= , J. Cell Biol. 123:637-652 (1993).