多肽合成技巧与标记技术.docVIP

多肽合成技巧与标记技术 ????多肽合成技巧??? ??? N末端的生物素标记 ??? ??? Wash 0.1 mmol resin with DMF. ??? Dissolve 0.244 g (+)-biotin (1 mmol, MW 244.3) in 5 mL DMF-DMSO (1:1) solution. A little warming is necessary. ??? Add 2.1 mL 0.45 M HBTU/HOBt solution and 0.3 mL DIEA to the solution prepared in step 2. 牋?Add the activated biotin solution to the resin and let stir overnight. 牋?Check resin to make sure coupling is complete as evidenced by negative ninhydrin test (colorless). 牋?Wash resin with DMF-DMSO (1:1) (2x) to remove excess (+)-biotin. 牋?Wash resin with DMF (2x) and DCM (2x). 牋?Let the resin dry before proceeding to cleavage. 牋?在2-氯TRT树脂上挂第一个FMOC氨基酸的方法 ??? ??? Weigh 10 g 2-chlorotrityl chloride resin (15 mmol) in a reaction vessel, wash with DMF (2x), swell the resin in 50 mL DMF for 10 min, drain vessel. ??? Weigh 10 mmol Fmoc-amino acid in a test tube, dissolve Fmoc-amino acid in 40 mL DMF, transfer the solution into the reaction vessel above, add 8.7 mL DIEA (50 mmol), swirl mixture for 30 min at room temperature. 牋?Add 5 mL methanol into the reaction vessel and swirl for 5 min. 牋?Drain and wash with DMF (5x). 牋?Check substitution. 牋?Add 50 mL 20% piperidine to remove the Fmoc group. Swirl mixture for 30 min. 牋?Wash with DMF (5x), DCM (2x), put resin on tissue paper over a foam pad and let dry at room temperature overnight under the hood. Cover the resin with another piece of tissue paper, press lightly to break aggregates. ??? Weigh loaded resin. ??? Pack in appropriate container. ??? 检测树脂上面是否挂上第一个FMOC氨基酸的方法 ??? ??? Weigh duplicate samples of 5 to 10 mg loaded resin in an eppendorf tube, add 1.00 mL 20% piperidine/DMF, shake for 20 min, centrifuge down the resin. 牋?Transfer 100 礚 of the above solution into a tube containing 10 mL DMF, mix well. 牋?Pipette 2 mL DMF into each of the two cells (reference cell and sample cell), set spectrophotometer to zero. Empty the sample cell, transfer 2 mL of the solution from step 2 into t