一、Introduction to Phycoerythrin

Phycoerythrin is isolated and purified from red algae. It emits intense fluorescence, possesses excellent light absorption properties and a high quantum yield, and has a broad excitation and emission range in the visible spectral region. It can be easily conjugated with biotin, avidin, and various monoclonal antibodies using conventional labeling methods to prepare fluorescent probes. These probes are used in diagnostic and bioengineering technologies such as fluorescent microscopic detection, fluorescent immunoassays, dual-color or multi-color fluorescence analysis, detection of antigens on the surface of cancer cells, fluorescent antibody labeling in flow cytometry, and in vivo imaging applications.

二、Experimental Materials

Phycoerythrin dye; PD-10 column (Sephadex G-25M gel filtration column), product of Cytiva, catalog No. 17-0851-01; NAP5 column (Sephadex G-25 DNA grade gel filtration column), product of Cytiva, catalog No. 17-0853-02; Succinimidyl-4-(N-methylmaleimide)cyclohexane-1-carboxylate (SMCC), product of Thermo, catalog No. 22360; N-ethylmaleimide (NEM), product of Thermo, catalog No. 23030; Dimethyl sulfoxide (DMSO), product of Thermo, catalog No. D12345.

三、Experimental Methods

1、Derivatization of Phycoerythrin

① Dissolve succinimidyl-4-(N-methylmaleimide)cyclohexane-1-carboxylate (SMCC) in anhydrous dimethyl sulfoxide (DMSO) to prepare a 10 mg/mL stock solution. React phycoerythrin (PE) with SMCC at a crosslinking molar ratio of 1:80. Seal with aluminum foil and incubate with rotation at room temperature for 60 minutes to allow derivatization via reaction between the amino groups on PE molecules and succinamide.

② Pre-equilibrate the gel filtration column with exchange buffer. Load the derivatized PE onto the column and collect the protein peak.

2、Antibody Treatment

① Dissolve dithiothreitol (DTT) in distilled water to prepare a 1 mol/L DTT stock solution. Adjust the antibody concentration to at least 4 mg/mL.

② Add 20 μL of DTT stock solution to each milliliter of antibody solution. Incubate at room temperature for 30 minutes to break the disulfide bonds of the antibody and form sulfhydryl groups.

③ Pre-equilibrate the gel column with exchange buffer. Load the reaction solution onto the column and collect the antibody fraction.

3、Covalent Crosslinking of Phycoerythrin and Antibody

① Add 3.2 mg of SMCC-derivatized PE to each milligram of antibody. Seal with aluminum foil and incubate with rotation at room temperature for 60 minutes to achieve covalent crosslinking between maleimide groups and sulfhydryl groups on the antibody.

② Dissolve 0 mg of N-ethylmaleimide (NEM) in 1.0 mL of anhydrous dimethyl sulfoxide (DMSO) to prepare NEM stock solution (prepared fresh before use).

③ Add 3.4 μL of NEM stock solution to each milligram of antibody. Seal with aluminum foil and incubate with rotation at room temperature for 60 minutes. After the reaction, the sulfhydryl groups on the antibody will be blocked.

4、Storage of Crosslinked Conjugates

Dialyze the crosslinked conjugates in storage buffer and store at 2–8°C.