The official Antibody labeling protocol provided with a protein labeling kit recommends using a 1 mg/mL concentration of the antibody for the efficient labeling. However, for some scarce antibodies and dilute samples, this concentration is too high and hard to achieve. Following the requests of our customers, we have evaluated the outcomes of labeling reactions when antibody concentration is 0.25 or 0.5 mg/mL. Different amounts of dye stock solution* (left column) were added to these dilute Ab solutions; the resulting Dye/Ab degree of labeling is listed in the table below.
| Reagent* volume, uL | Ab concentration = 0.25 mg/mL | Ab concentration = 0.5 mg/mL |
| 2 | 0.70 | - |
| 3.5 | 1.15 | - |
| 5 | 1.54 | 1.18 |
| 7 | 2.20 | 1.95 |
| 10 (or add Ab solution to dye) | 3.15 | 2.20 |
The reaction was performed at 25°C with 100 uL of Ab solution for 30 min. So, when the antibody to be labeled is dissolved at 0.25 mg/mL, we recommend to use 7 uL of dye solution per 100 uL of Ab solution to achieve a Dye/Ab ratio of 2.2. When the antibody is dissolved at 0.5 mg/mL (i.e., 50 ug / 100 uL), we recommend to to use a complete vial of the dye per 100 uL of Ab solution. This can be achieved either by dissolving the dye in 10 uL of anhydrous DMSO or just by adding Ab solution to the dye vial without a co-solvent.
* Dye stock solution is prepared by dissolution of dye in 10 uL of DMSO - please refer to the protocol.