Labelling myosin antibody

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Revision as of 08:20, 16 February 2015 by Stephanh (talk | contribs)
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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.

Concentration:

  1. Equilibrate NAP-5 with 0.2M Borate buffer
  2. Add 500 µl of myosin antibody to column
  3. Elute with 1ml borate buffer
  4. If necessary concentrate antibody to 1-3 mg/ml
  5. 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.