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Antibody Labeling with HRP
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Flash content link: https://vlab.amrita.edu/repo/BIOTECH/IMM/AntIbody_%20labeling_%20with%20_HRP/index.swf

 

Materials required

 

  • Weighing machine
  • Blotting paper
  • Microfuge tube
  • Micropipette
  • Pipette
  • Pipette pump
  • Dialysis tubing membrane
  • Forceps
  • Wash bottle
  • Dialysis clamps
  • Magnetic stirrer
  • Stir bar
  • Visicooler
  • Pastuer pipette
  • Cryo vial
  • -20o C freezer
  • Cryo box
  • Glass rod
  • 5ml test tube
  • Centrifuge tube rack
  • Gel filtration coloum
  • Cuvetttes
  • UV spectrophotometer

 

 Reagents required

 

  •  Horse radish peroxidase
  •  Sodium periodate solution
  •  Ethanol storage solution
  •  1mM Sodium acetate buffer
  •  0.2M Sodium carbonate buffer
  •  Sodium borohydride solution
  •  Borate buffer
  •  50% Glycerol
  •  PBS(Phosphate buffered saline) buffer
  •  Sepharose CL-6B

 

Procedure

 

1.Dissolve 4mg of peroxidase in 1mL of water.

2. Add 200 µL of freshly prepared 0.1 M sodium periodate, and stir the solution for 20 min at
room temperature.

3. Dialyze the modified enzyme against 1mM sodium acetate buffer (pH 4.4) overnight
at 4oC.

5. Adjust the pH of the dialyzed enzyme solution by adding 20 µL of 0.2 M sodium carbonate buffer (pH 9.5) and add 1ml of IgG solution. Stir the mixture for 2 h at room temperature.

6. Add 100 µL of freshly prepared Sodium borohydride solution (4 mg/mL), and stir the
mixture occasionally over a period of 2 h at 4oC.

7. Fractionate the mixture by gel filtration on a column of Sepharose CL-6B in PBS and determine the A280 and A403.

8. Pool the fractions in the first peak (both A280 and A403 peaks coincide) and stored at -20 oC with Glycerol added at a final concentration of 50%.

 

 

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