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3x DYKDDDDK Tag是研究无法获得抗体(Abs)的蛋白质的功能和定位的有用工具。
编号:205011
CAS号:921765-76-6
单字母:H2N-DYKDDDDKDYKDDDDKDYKDDDDK-OH
| 溶解度: | DMSO : 100 mg/mL (33.30 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO) |
3x DYKDDDDK标签是一种有用的工具,用于研究其抗体(Abs)不可用的蛋白质的功能和定位。它也常以3X FLAG格式(3x DYKDDDDK标签)用于纯化低丰度积累的难纯化蛋白质。
3x DYKDDDDK Tag is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. Often it is also used in a 3X FLAG format (3x DYKDDDDK Tag) for purifying difficult proteins that accumulate in low abundance .
收获瞬时转染了表达HARS质粒的HEK293细胞,并在含有哺乳动物蛋白酶抑制剂鸡尾酒的CelLytic M缓冲液中裂解20分钟,裂解温度为4°C。根据制造商的协议,通过与抗DYDDDDK树脂结合来纯化FLAG标记的HARS,并在含有50 mM Tris-HCl(pH 7.4)和150 mM NaCl的缓冲液中,通过与3X-DYKDDDDK肽竞争进行洗脱(人HARS的纯化)[2]。
HEK293 cells transiently transfected with HARS-expressing plasmids were harvested and lysed in CelLytic M buffer containing mammalian protease inhibitor cocktail for 20 min at 4 °C. FLAG-tagged HARS is purified by binding to an anti-DYDDDDK resin per manufacturer protocol and eluted by competition with 3X-DYKDDDDK peptide in buffer containing 50 mM Tris-HCl pH 7.4, and 150 mM NaCl (purification of human HARS)[2].
References:
[1]. Mishra V. Affinity Tags for Protein Purification. Curr Protein Pept Sci. 2020;21(8):821-830.
[2]. Abbott JA, et al. The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity. Biochemistry. 2017;56(28):3619-3631.
3xFlag DYKDDDDK, supplied as the ammonium salt
专肽生物可以提供常见抗体制备、免疫学研究等生物科研需求的各类标签多肽(Tag peptide)以及蛋白底物产品,可用荧光标记(FITC、5-Fam)等。除常见抗体中抗原决定部位的标签多肽(tag: His | c-Myc | HA | FLAG | V5 | X-press | VSV tag | T7 tag | Snoop tag | E-tag | Sof tag 1 | Spy tag |等等),我们还提供各类定制标签肽的合成,并可提供荧光标记(FITC、5-Fam)等。同时,免费提供的MS和HPLC分析报告。




















多肽H2N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH的合成步骤:
1、合成CTC树脂:称取1.69g CTC Resin(如初始取代度约为1.19mmol/g)和2.41mmol Fmoc-Lys(Boc)-OH于反应器中,加入适量DCM溶解氨基酸(需要注意,此时CTC树脂体积会增大好几倍,避免DCM溶液过少),再加入6.03mmol DIPEA(Mw:129.1,d:0.740g/ml),反应2-3小时后,可不抽滤溶液,直接加入1ml的HPLC级甲醇,封端半小时。依次用DMF洗涤2次,甲醇洗涤1次,DCM洗涤一次,甲醇洗涤一次,DCM洗涤一次,DMF洗涤2次(这里使用甲醇和DCM交替洗涤,是为了更好地去除其他溶质,有利于后续反应)。得到 Fmoc-Lys(Boc)-CTC Resin。结构图如下:

2、脱Fmoc:加3倍树脂体积的20%Pip/DMF溶液,鼓氮气30分钟,然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Lys(Boc)-CTC Resin 。(此步骤脱除Fmoc基团,茚三酮检测为蓝色,Pip为哌啶)。结构图如下:

3、缩合:取6.03mmol Fmoc-Asp(OtBu)-OH 氨基酸,加入到上述树脂里,加适当DMF溶解氨基酸,再依次加入12.07mmol DIPEA,5.73mmol HBTU。反应30分钟后,取小样洗涤,茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。(洗涤树脂,去掉残留溶剂,为下一步反应做准备)。得到Fmoc-Asp(OtBu)-Lys(Boc)-CTC Resin。氨基酸:DIPEA:HBTU:树脂=3:6:2.85:1(摩尔比)。结构图如下:

4、依次循环步骤二、步骤三,依次得到
H2N-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
H2N-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
Fmoc-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin
以上中间结构,均可在专肽生物多肽计算器-多肽结构计算器中,一键画出。
最后再经过步骤二得到 H2N-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-Asp(OtBu)-Tyr(tBu)-Lys(Boc)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Asp(OtBu)-Lys(Boc)-CTC Resin,结构如下:

5、切割:6倍树脂体积的切割液(或每1g树脂加8ml左右的切割液),摇床摇晃 2小时,过滤掉树脂,用冰无水乙醚沉淀滤液,并用冰无水乙醚洗涤沉淀物3次,最后将沉淀物放真空干燥釜中,常温干燥24小试,得到粗品H2N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH。结构图见产品结构图。
切割液选择:1)TFA:H2O=95%:5%
2)TFA:H2O:TIS=95%:2.5%:2.5%
3)三氟乙酸:茴香硫醚:1,2-乙二硫醇:苯酚:水=87.5%:5%:2.5%:2.5%:2.5%
(前两种适合没有容易氧化的氨基酸,例如Trp、Cys、Met。第三种适合几乎所有的序列。)
6、纯化冻干:使用液相色谱纯化,收集目标峰液体,进行冻干,获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。
7、最后总结:
杭州专肽生物技术有限公司(ALLPEPTIDE https://www.allpeptide.com)主营定制多肽合成业务,提供各类长肽,短肽,环肽,提供各类修饰肽,如:荧光标记修饰(CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等),功能基团修饰肽(叠氮、炔基、DBCO、DOTA、NOTA等),同位素标记肽(N15、C13),订书肽(Stapled Peptide),脂肪酸修饰肽(Pal、Myr、Ste),磷酸化修饰肽(P-Ser、P-Thr、P-Tyr),环肽(酰胺键环肽、一对或者多对二硫键环),生物素标记肽,PEG修饰肽,甲基化修饰肽等。
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