Peridinin-Chlorophyll-Protein (PerCP) is a peridinin-chlorophyll-protein complex with a molecular weight of 35.5 kDa, isolated from dinoflagellates (Dinophyceae sp.). It has a broad excitation spectrum with a main peak at 482 nm, a large Stokes shift, and an emission peak at 677 nm. PerCP and its conjugates are large complexes suitable for cell surface labeling techniques such as flow cytometry. PerCP-Cy5.5 is a stable tandem dye excited by 488 nm laser. It can stain low-abundance targets. In multicolor flow cytometers, the Cyanine5.5 signal can usually be detected stably. When detected together with APC on dual-laser tube instruments, compensation adjustment is required, but less than that for PerCP.
Product Properties
Purity: A482/A280 > 4.2
Excitation Wavelength: 480 nm
Emission Wavelength: 697 nm
Form: Solution
Stability and Storage
Storage Conditions: Store at 2-8°C in the dark, do not freeze.
Stability: Can be stored for at least 6 months under suitable conditions.
Usage Methods
1. Antibody Modification
① Dissolve antibody modification reagent (DTT/TCEP) in ddH₂O to prepare an antibody modification solution with a concentration of 2 mg/ml.
② Take the antibody to be labeled (purity > 90%), adjust its concentration to approximately 5-10 mg/ml with PBS buffer, add 10 μl of the antibody modification solution per mg of antibody, mix gently, and stir at room temperature for 60-90 minutes.
③ After the reaction is completed, transfer to the inner core of an ultrafiltration tube, add MES buffer, and remove excess antibody modification reagent by repeated centrifugation and solution replacement through the ultrafiltration tube. The collected solution in the inner core of the ultrafiltration tube is the modified antibody, and adjust its concentration to 1-5 mg/ml.
2. Activation of PerCP-Cy5.5 Tandem Dye
① Dissolve PerCP-Cy5.5 tandem dye in 0.1M PBS (pH=7.4, 5mM EDTA) to a concentration of 5-10 mg/ml.
② Dissolve SMCC in anhydrous dimethyl sulfoxide (DMSO) to prepare a mother liquor with a concentration of 10 mg/ml.
③ Add 18 μl of SMCC per mg of PerCP-Cy5.5 tandem dye (n(PerCP-Cy5.5): n(SMCC) = 1:20), seal with aluminum foil, and react at room temperature with rotation for 1 hour to allow the amino groups on the PerCP-Cy5.5 tandem dye molecules to react with succinamide to form derivatized PerCP-Cy5.5 tandem dye.
④ After the reaction is completed, transfer the reaction solution to an ultrafiltration centrifuge tube, centrifuge at 4°C, 12000 g for 5 minutes, remove the filtrate, add 500 μl of MES buffer, mix well and centrifuge; repeat this operation 5 times.
⑤ Collect the activated PerCP-Cy5.5 tandem dye solution.
3. Conjugation of Activated PerCP-Cy5.5 with Antibody
① Adjust the concentration of activated PerCP-Cy5.5 to 5 mg/ml with MES buffer; to obtain the accurate weight of PerCP-Cy5.5, it is recommended to use the extinction coefficient of PerCP for measurement (i.e., [PerCP-Cy5.5] = 0.086 × A482, where [PerCP-Cy5.5] is the concentration in mg/ml, A482 is the absorbance at a wavelength of 482 nm, and the control range is 0.3 ~ 0.8).② Mix the modified antibody and activated PerCP-Cy5.5 at a mass ratio of 2.25:1 (0.45 mg of activated PerCP-Cy5.5 per mg of modified antibody), and react at room temperature in the dark for 2 hours.
③ Dissolve NEM in anhydrous dimethyl sulfoxide (DMSO) to prepare a 12.5 mg/ml solution (0.1M), add NEM solution at a volume of 5 μl per mg of antibody to the reaction product in step ② to block unreacted active groups.
④ Add the solution from step ③ to an ultrafiltration tube and remove blocking reagents, etc., by repeated centrifugation.
⑤ Aliquot the labeled antibody, add an appropriate protectant, and store at -20°C for later use.
Precautions
1. PerCP-Cy5.5 needs to be stored in the dark at low temperature, and try to ensure it is protected from light during the labeling process.
2. Blocking agents and modifiers should be prepared and used immediately and cannot be stored for a long time.
3. The labeled antibody should have high specificity and a purity of not less than 90%. It is best to use monoclonal antibodies, and the solution environment should not contain free amino groups, preferably PBS solution. Before labeling, NaN3 and BSA should be removed from the antibody. Operations such as dialysis, concentration, and concentration determination of the antibody will cause loss of antibody quantity. Therefore, when preparing the antibody before labeling, the optimal amount of antibody should be considered according to specific circumstances.
4. Since the groups carried by the antibody after modification are easily re-oxidized, the modified antibody should be conjugated with Activated PerCP-Cy5.5 as soon as possible.
