Labelling myosin antibody

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test myotube cells

Material

  • 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.