Labelling myosin antibody

test myotube cells

Materials

• 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

Procedure

1. Measure concentration of myosin antibody using Biorad reagent and rabbit IgG as standard
2. Equilibrate NAP-5 with 0.2M Borate buffer
3. Add 500 µl of myosin antibody to column
4. Elute with 1ml borate buffer
5. If necessary concentrate antibody to 1-3 mg/ml
6. Add 25 µl of FLNHS per 500 µl of antibody
7. Incubate 4 hrs at room temp
8. While waiting, equilibrate NAP-10 with PBS/azide
9. Quench with 30 µl of 100 mM glycine per 500 µl
10. Load antibody onto Nap-10 column: (1 ml total)
11. Elute with 1.5 ml PBS/azide
12. Add 150 µl of 1% BSA
13. Store in fridge and test cells at 1:20, 1:50, 1:100, 1:200.
14. Long-term storage: Add 50% v/v glycerol and store at –20 C.