Labelling myosin antibody

0.2 M Borate buffer, pH 8 Biorad protein assay substance IgG standard Anti myosin antibody (4A1025) FlNHS (1.5 mg/ml in DMSO—make up fresh just before use) 100mM glycine, pH 7 1%BSA in PBS azide NAP-5 NAP-10 PBS azide (test myotube cells)

1 Measure concentration of myosin antibody using Biorad reagent and rabbit IgG as standard.

Concentration: 2 Equilibrate NAP-5 with 0.2M Borate buffer 3 Add 500 µl of myosin antibody to column 4 5 Elute with 1ml borate buffer 6 If necessary concentrate antibody to 1-3 mg/ml 7 Add 25 µl of FLNHS per 500 µl of antobody Added: 8 Incubate 4 hrs at room temp 9 While waiting, equilibrate NAP-10 with PBS/azide 10 Quench with 30 µl of 100 mM glycine per 500 µl Added: 10 minutes 11 Load antibody onto Nap-10 column: (1 ml total)

12 Elute with 1.5 ml PBS/azide 13 Add 150 µl of 1% BSA 14 Store in fridge and test cells at 1:20, 1:50, 1:100, 1:200. 15 Long-term storage: Add 50% v/v glycerol and store at –20 C.