Coupling of antibody with peroxidase

 

Theory:       

 

*         Peroxidase is a glycoprotein und thus can be oxidized forming aldehyde groups.

*         ABs couple with their respective amino groups forming imines.     

*         To stabilize the bonds the imines are reduced to amines.

 

Protocol:    

 

1.      Dissolve 4 mg HRP(Peroxidase) in 1ml Aqua bidest.

 

2.      + 240 µl 0.1 M sodium perjodate in 10 mM PBS, pH = 7.0

 

3.      20 min , RT

 

4.      Dialysis with 1mM sodium acetate buffer, pH = 4.0, 24 h

 

5.      Parallel dialysis of 400 µl AB (10 mg/ml) with

         sodium carbonate / bicarbonate-buffer, pH = 9.5, 24h

 

6.      Mix AB and activated peroxidase

 

7.      2h, RT

 

8.      + 100 µl sodium borohydride (4 mg/ml) in aqua bidest.

 

9.      2h, 4°C

 

10.     Dialysis with PBS, pH = 7.4, 24 h

 

11.    Store at 4°C with 0.1% sodium azide