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.
Add the activated biotin solution to the resin and let stir overnight.
Check resin to make sure coupling is complete as evidenced by negative ninhydrin test (colorless).
Wash resin with DMF-DMSO (1:1) (2x) to remove excess (+)-biotin.
Wash resin with DMF (2x) and DCM (2x).
Let the resin dry before proceeding to cleavage.
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.
Add 5 mL methanol into the reaction vessel and swirl for 5 min.
Drain and wash with DMF (5x).
Check substitution.
Add 50 mL 20% piperidine to remove the Fmoc group. Swirl mixture for 30 min.
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.
Transfer 100 µL of the above solution into a tube containing 10 mL DMF, mix well.
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 the sample cell, check absorbance.
Subs = 101(A)/7.8(w)
A = absorbance
w = mg of resin
Check absorbance three times at 301 nm, calculate average substitution.
FMOC方法合成多肽操作步骤 (0.25 mmol)
Wash resin with DMF (4x) and then drain completely.
Add approximately 10 mL 20% piperidine/DMF to resin. Shake for one min and drain.
Add another 10 mL 20% piperidine/DMF. Shake for 30 min.
Drain reaction vessel and wash resin with DMF (4x). Make sure there is no piperidine remaining. Check beads using ninhydrin test, beads should be blue.
Coupling Step - Prepare the following solution:
1 mmol Fmoc-amino acid
2.1 mL 0.45 M HBTU/HOBT (1mmol)
348 µL DIEA (2 mmol)
Add above solution to the resin and shake for a minimum of 30 min. This coupling step can be longer if desired.
Drain reaction vessel and wash resin with DMF (4x).
Perform Ninhydrin test:
If negative (colorless), proceed to step 2 and continue synthesis.
If positive (blue), return to step 5 and re-couple the same Fmoc-amino acid. Increase the coupling time if necessary.
合成含有磷酸化酪氨酸多肽的方法
Reagent: N-a-Fmoc-O-phosphotyrosine
For 0.1 mmol or 0.25 mmol synthesis, use 0.483 g Fmoc-Tyr(PO3H2)-OH (1 mmol, MW 483.4) . For ABI synthesizers, pack Fmoc-Tyr(PO3H2)-OH in a cartridge.
The cycle program for coupling Fmoc-Tyr (PO3H2)-OH is the same as for other Fmoc-amino acids except for the coupling time (see step 3). (Note: ABI synthesizers use HBTU/HOBT as the activating reagent.)
The coupling time for Fmoc-Tyr(PO3H2)-OH needs to be increased. For ABI model 430A peptide synthesizer, insert several steps (i.e., vortex on, wait 990 sec, vortex off, to increase the coupling time). For ABI model 431A peptide synthesizer, add additional "I"s. Overnight coupling may be necessary for some sequences.
After the coupling step for Fmoc-Tyr(PO3H2)-OH, perform ninhydrin test to ensure complete coupling. Negative (colorless) ninhydrin test indicates complete coupling, while a positive (blue) ninhydrin test indicates incomplete coupling.
Increase the coupling time of the amino acid residues after
一：公司介绍
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二：我们的专长
（1）普通线性肽，链长至120个氨基酸，毫克至克级，纯度可达99%
（2）简单修饰肽：免费提供N端乙酰化，末端酰胺化
（3）C端：酰胺化（NH2），生物素标记 (用赖氨酸侧链连接)，PEG2(用赖氨酸侧链连接)，
C-FITC(用赖氨酸侧链连接)，C-AMC
N端：乙酰化（AC），生物素标记（Biotin），脂肪酸修饰(Pal, Myr)，罗丹明标记，
898995850
