Antibody conjugation with  Biotin or Alexa fluorescent dyes

 

Materials

1)      antibody  that will be coujgated

2)       Biotin cross-linker (Pierce Cat.No = 21343)

3)       Alexa fluorescent dyes ( Molecular Probe Cat.No :  Alexa Fluor 350 (blue) =  A20801

                                                                                                               488 (green) = A20181

                                                                                                                568 (red)  =  A20184

4)      Centricon YM-50 (for easily purifying conjugated antibodies)

Amicon Cat No. =  4224  (for filtering IgG above Molecular Weight 50,000)

* you can get another Molecular Weight filter system….ex….> MW100,000

 

You can easily conjugate your antibodies following simple method in the respective protocols…

I haven’t try Alexa series….but also it’s simple….just try….

I here describe the simple protocol with Biotin-cross linker that I have tried……

 

22D1 biotinylation w/ NHS-Biotin (Molecul Probe , Pierce Cat.No = 21343)

1)    prepare 2mg of 22D1 with 1ml cold 1X PBS in E.tube

2)   add 0.2mg of NHS-Biotin ( Stock = 100mg/ml of DMSO)

3)   rotating   E.tube 2hr at 4C (or 30min at Room temp.)

5)      removing the unreated NHS-Biotin by washing with 1X cold PBS

–          transfer the sol to Centricon (MW50,000)

–          mass up to 2ml with 1x cold PBS

–          centrifuge 1900rpm, about 30 min, remaining 200-300ul….

–          Washig one more time with same procedure

–          Concentrating to the volule (200-500ul) that I wish

6)      Biotinylation check with Western or FACS etc…..using tissue or transfectants cell lines etc….

Ex. DCEK-DC-SIGN ( negative control)    +    Biotin-22D1   =   incubation 1hr at 4C

       DCEK-SIGN-R1

 

      —–   staining with StreptoAvidin*FITC

      —–   FACS reading….

 

The End….

So easy….isn’t it…..????

If  you have any questions, just let me know…..