Eye Pad,Breathable Non-Woven Mash Eye Pad,Cotton Breathable Mesh Eye Pad,Medical Absorbent Eye Pad Shaoxing Weijian Medical Supplies Co.,Ltd. , https://www.wetourmedical.com
Method for phycoerythrin labeling antibody & PE-labeled protein A method
Method for phycoerythrin labeling antibody & PE-labeled protein A method
       Â
Method of phycoerythrin labeled antibody:
1. Â Â Â Â Â Preparation of thiolated phycoerythrin (PE);
(1)         600 μl of a concentration of 15.5 mg/ml of sterol hydrochloride to 1.2 ml of PE at a concentration of 3.6 mg/ml;
(2)         The above mixture was mixed with 1.2 ml of PB pH 6.8, and then placed in a dialysis bag and placed in 50 mmol/L PB of pH 6.8 and dialyzed at 4 ° C overnight;
(3) Â Â Â Â Â Â Â Â Then exchanged with pH 7.5 PB for 6h. 8 thiol groups can be combined in each PE molecule;
2. Â Â Â Â Â Preparation of sulfhydryl PE-IgG;
(1)         30 μl of a 1.1 mg/ml solution of a heterobifunctional reagent SPDP in ethanol was added to 700 μl of a 4.2 mg/ml IgG PB solution (50 mmol/L pH 7.5), and reacted at room temperature for 2.5 h;
(2)         400 μl of 1.7 mg / ml of thiolated PE was added to 500 μl of the reaction mixture, and reacted at room temperature for 12 h;
(3)         100 μl of 50 mmol/L sodium iodoacetate was added to block the residual sulfhydryl group, and dialyzed overnight at 4 ° C with PB;
(4)         Finally, 0.01% Na 3 N 3 was added and then packaged and stored at 4 ° C for half a year;
PE- labeled protein A method:
1.      Weigh 4.08mg of PE and dissolve it in 1ml of PBS with a concentration of 0.1mmol/ml pH7.4. After dissolving, remove 0.5ml, add 10μl of SPDP anhydrous methanol solution with a concentration of 2.65mg/ml, and the amount of SPDP/protein substance. The reaction was carried out at 10,22 ° C for 5 min, passed through Sephadex G-50 (1×17 cm), and equilibrated and eluted with 100 mmol/L PBS pH 7.4;
2.      0.5 ml of 2 mg/ml protein and 100 mmol/L pH 7.4 PBS were mixed, and 2.6 μl of the above SPDP methanol solution was added to the mixture so that SPDP: protein = 9:5, after being placed at 22 ° C for 40 min. Adding 25 μl of dithiothreitol (DTT) buffer of pH 7.4, reacting at 22 ° C for 25 min, passing Sephadex G-50, collecting protein A peak;
3.      The 0.77 mg/ml PE and the 0.27 mg/ml protein A were mixed in equal amounts, and after reacting at 22 ° C for 6 h, the mixture was stored at 4 ° C for use;
4.      The above two PE labeled products were dissolved in 0.01 mol/L pH 7.4 PB (containing 0.1 mol/L DETA, 1 mol/L iodoacetamide, 1% BSA and 0.1% NaN 3 ), and stored at 0-4 ° C;