Difference between revisions of "Labelling myosin antibody"
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=== procedure: === | === procedure: === | ||
| − | # Measure concentration of myosin antibody using Biorad reagent and rabbit IgG as standard | + | # Measure concentration of myosin antibody using Biorad reagent and rabbit IgG as standard |
| − | |||
| − | |||
# Equilibrate NAP-5 with 0.2M Borate buffer | # Equilibrate NAP-5 with 0.2M Borate buffer | ||
# Add 500 µl of myosin antibody to column | # Add 500 µl of myosin antibody to column | ||
# Elute with 1ml borate buffer | # Elute with 1ml borate buffer | ||
# If necessary concentrate antibody to 1-3 mg/ml | # If necessary concentrate antibody to 1-3 mg/ml | ||
| − | # Add 25 µl of FLNHS per 500 µl of | + | # Add 25 µl of FLNHS per 500 µl of antibody |
| − | + | # Incubate 4 hrs at room temp | |
| − | + | # While waiting, equilibrate NAP-10 with PBS/azide | |
| − | + | # Quench with 30 µl of 100 mM glycine per 500 µl | |
| − | + | # Load antibody onto Nap-10 column: (1 ml total) | |
| − | + | # Elute with 1.5 ml PBS/azide | |
| − | + | # Add 150 µl of 1% BSA | |
| − | + | # Store in fridge and test cells at 1:20, 1:50, 1:100, 1:200. | |
| − | + | # Long-term storage: Add 50% v/v glycerol and store at –20 C. | |
| − | |||
| − | |||
| − | |||
| − | |||
Revision as of 08:28, 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:
- Measure concentration of myosin antibody using Biorad reagent and rabbit IgG as standard
- Equilibrate NAP-5 with 0.2M Borate buffer
- Add 500 µl of myosin antibody to column
- Elute with 1ml borate buffer
- If necessary concentrate antibody to 1-3 mg/ml
- Add 25 µl of FLNHS per 500 µl of antibody
- Incubate 4 hrs at room temp
- While waiting, equilibrate NAP-10 with PBS/azide
- Quench with 30 µl of 100 mM glycine per 500 µl
- Load antibody onto Nap-10 column: (1 ml total)
- Elute with 1.5 ml PBS/azide
- Add 150 µl of 1% BSA
- Store in fridge and test cells at 1:20, 1:50, 1:100, 1:200.
- Long-term storage: Add 50% v/v glycerol and store at –20 C.