Labelling myosin antibody: Difference between revisions
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=== material: === | === material: === | ||
0.2 M Borate buffer, pH 8 | * 0.2 M Borate buffer, pH 8 | ||
Biorad protein assay substance | * Biorad protein assay substance | ||
IgG standard | * IgG standard | ||
Anti myosin antibody (4A1025) | * Anti myosin antibody (4A1025) | ||
FlNHS (1.5 mg/ml in DMSO—make up fresh just before use) | * FlNHS (1.5 mg/ml in DMSO—make up fresh just before use) | ||
100mM glycine, pH 7 | * 100mM glycine, pH 7 | ||
1%BSA in PBS azide | * 1%BSA in PBS azide | ||
NAP-5 | * NAP-5 | ||
NAP-10 | * NAP-10 | ||
PBS azide | * PBS azide | ||
=== procedure: === | === procedure: === | ||
Revision as of 08:18, 16 February 2015
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: 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.