Conjugation Troubleshooting

My conjugation didn't work. What do I do now?


Contact us. In order to enable us to respond effectively, please answer the following questions and fax or e-mail them to us.


  1. What product did you use for the conjugation? (Please provide product code and lot number.)
  2. How did the product specifically disappoint; i.e. low signal, poor conjugation, high precipitation?
  3. What mass ratio of activated phycobiliprotein (SMCC xlAPC or SMCC RPE) to antibody was used? (Each kit booklet provides a recommended ratio which is generally applicable to antibody conjugations, but any given antibody may conjugate best at a different ratio.)
  4. What kind of Ab was it (MAb, polyclonal, source, class, subclass), or was it some other protein?
  5. What was the starting quantity and concentration of the Ab?
  6. What buffer was the Ab in initially?
  7. After reducing the Ab, what volume was put on desalting column A (or Spin Column D) and what volume was collected?
  8. Did any precipitate form following the mixing of the reduced Ab and the activated phycobiliprotein? How much?
  9. (optional) Can you spin the "failed" Ab-phycobiliprotein conjugate and provide the absorbances of the supernatant at 280 nm and 650 nm (APC) or 566 nm (RPE)?
  10. Is there anything else we ought to know?