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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…..
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