Difference between revisions of "Labelling myosin antibody"
Jump to navigation
Jump to search
| (5 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
| − | test myotube cells | + | ''test myotube cells'' |
| − | == | + | == 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 == |
| − | + | # 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. | |
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
Latest revision as of 08:49, 19 June 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.