Frequently Asked Questions

The standard reaction size for your PhycoLink APC (PJ25K) and RPE (PJ31K) Conjugation Kits is 1 mg of antibody. Can I conjugate four each of 0.25 mg of antibody instead? How about 10 each of 0.1 mg of antibody?

Yes. For the 0.25 mg of antibody, scale down all of the components in the conjugation accordingly. The antibody coming off the desalting column will be more dilute, so the conjugation reaction will take place at lower concentrations. In most cases, this poses no problem.

Please note that when using the kit components for multiple reactions, be sure to flush each desalting column with several column volumes of the appropriate buffer before each additional reaction (see the appendix in the package insert for buffer recipes).

We successfully conjugated as little as 0.1 mg of antibody with both the APC and RPE Conjugation Kits. For 0.1 mg of antibody this protocol was used:

1. 20 µL of DTT stock was added to 1 mL of 0.1 mg/ml antibody solution and incubated for 30 minutes at room temperature. Higher concentrations of antibody may be preferable. We have intentionally used a dilute solution of antibody to test the limits of the conjugation. For smaller volumes of antibody, use correspondingly smaller volumes of DTT stock.
2. The reduced antibody was applied to a desalting column equilibrated with Exchange Buffer (Column A) and antibody containing fractions were eluted from the column and pooled. In this instance the volume of the pooled fractions was 2.5 mL. We intentionally used a large volume (i.e. low antibody concentration) to test the limits of the conjugation protocol. You might wish to pool a smaller volume and maintain a higher concentration of antibody in the subsequent conjugation.
3. Either 30 µL of the PhycoLink^® SMCC-APC (0.3 mg) or 30 µL of SMCC-RPE (0.3 mg) was added to the reduced antibody solution and mixed. The mixture was incubated for 2 hours at room temperature in the dark.
4. 2 µL of NEM Stock was added to the mixture followed by a 30-minute incubation at room temperature in the dark.
5. The conjugates were analyzed and compared to the results of a conjugation using 1 mg of antibody following the standard kit procedures:

Antibody Human IgG Phycobiliprotein % Soluble Conjugate* % Phycobiliprotein Incorporated** 0.1 mg 0.3 mg SMCC-APC 30 57 1.0 mg 3.0 mg SMCC-APC 42.3 61 0.1 mg 0.3 mg SMCC-RPE 28 41 1.0 mg 3.0 mg SMCC-RPE 46 62

* Percentage of total phycobiliprotein used in the conjugation which is incorporated into soluble conjugate. This value is measured by protein G-agarose binding.

** Percentage of total phycobiliprotein used in the conjugation which is incorporated into both soluble and insoluble conjugate (precipitate).

RESULTS

We conclude that as little as 0.1 mg of antibody at a starting concentration of 0.1 mg/mL can be successfully conjugated using the PhycoLink Conjugation Kits. Conjugation of 0.1 mg of antibody gives a modest decrease in the yield of usable conjugate under more dilute conditions compared to the standard conditions with both SMCC-APC and SMCC-RPE. Of course, actual results will vary wit individual antibodies.