CY3 labeling reaction principle and instructions for use

CY3 labeling reaction principle:
Reactive CY3 specifically reacts with primary amines (N-terminal and lysine residue side chains) to form stable amide bonds

Operation process:
Preparation before the experiment:
  1. Read the instruction manual carefully.
  2. Calculate the amount of Reactive CY3 to be used.
  3. Remove the kit from the refrigerator 20 minutes in advance and equilibrate to room temperature (Note: Reactive CY3 is not required to continue to be placed in the refrigerator).
  4. Dissolve Reactive CY3: Add 20 μL of DMF to Reactive CY3 bottle and let stand for 10 min until it is fully dissolved. At this time, the concentration of CY3 is 10 mM.

Operation steps : (This procedure is marked according to the amount of 1 mg)
  1. Take 1 mg of the antibody to be labeled in a Filtration tube, and add a corresponding volume of Labeling Buffer to a total volume of 0.5 mL and centrifuge at 12,000 × g for 10 min. (Note: 1Filtration tube has a maximum volume of 0.5 mL. 2 When the concentration of the antibody to be labeled is low, it can be centrifuged once by ultrafiltration.)
  2. Add 13.3 μL of Reactive CY3 and the appropriate amount of Labeling Buffer to the above Filtration tube to give a final antibody concentration of 2 mg/mL and mix gently by pipetting. Incubate in a 37 ° C incubator for 60 min in the dark.
  3. Centrifuge at 12,000 × g for 10 min.
  4. Add appropriate amount of Labeling Buffer to the above Filtration tube to make a final volume of 0.5 mL, mix gently by pipetting, and centrifuge at 12,000 × g for 10 min. And repeat the operation once.
  5. Add 0.2 mL of Labeling Buffer to the Filtration tube and gently blow. The filter element was inverted and placed in another centrifuge tube and centrifuged at 6,000 x g for 10 min.
  6. The solution in the centrifuge tube is collected as a CY3-labeled antibody.
Note :
  1. The kit may also label other antigens, HRPs, and polypeptides containing amino (NH2-), and the specific labeling ratio is determined according to the amount of amino groups in the label to be labeled.
  2. DMF should be sealed and stored dry, and sealed with a sealing film immediately after use.
  3. In the step 5, the Labeling Buffer can be used as a buffer for recovering the labeled antibody, and any other buffer or protective agent can also be used.
  4. This kit is valid for one year before it is opened. Please use it within the validity period.
  5. This kit provides a 10 kDa Filtration tube. Please note the molecular weight of the labeled antigen or peptide.
  6. In step 2, when labeling other antibodies, we should first control the final concentration of the antibody to 2 mg / mL, and then calculate the volume of Reactive CY3 should be added according to the amount of antibody.
  7. To determine that no free amino groups (Tris, ammonia and sodium azide are introduced in the coupling)

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