Labelling myosin antibody: Difference between revisions

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''test myotube cells''
''test myotube cells''


=== material: ===
== Material ==
* 0.2 M Borate buffer, pH 8
* 0.2 M Borate buffer, pH 8
* Biorad protein assay substance
* Biorad protein assay substance
Line 13: Line 13:
* PBS azide
* PBS azide


=== procedure: ===
== Procedure ==


1 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
Concentration:
# Add 500 µl of myosin antibody to column
2 Equilibrate NAP-5 with 0.2M Borate buffer
# Elute with 1ml borate buffer
3 Add 500 µl of myosin antibody to column
# If necessary concentrate antibody to 1-3 mg/ml
4
# Add 25 µl of FLNHS per 500 µl  of antibody
5 Elute with 1ml borate buffer
# Incubate 4 hrs at room temp
6 If necessary concentrate antibody to 1-3 mg/ml
# While waiting, equilibrate NAP-10 with PBS/azide
7 Add 25 µl of FLNHS per 500 µl  of antobody
# Quench with 30 µl of 100 mM glycine per 500 µl
Added:
# Load antibody onto Nap-10 column: (1 ml total)
8 Incubate 4 hrs at room temp
# Elute with 1.5 ml PBS/azide
9 While waiting, equilibrate NAP-10 with PBS/azide
# Add 150 µl of 1% BSA
10 Quench with 30 µl of 100 mM glycine per 500 µl
# Store in fridge and test  cells at 1:20, 1:50, 1:100, 1:200.
Added:
# Long-term storage:  Add 50% v/v glycerol and store at –20 C.
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.

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

  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.
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