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KRAS靶点难成药?-整体解决方案来帮您

更新时间:2023-11-21

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KRAS热度已经消退了?NO...据调查,现在除传统针对KRAS靶点小分子药物外,更多玩家尝试新兴疗法征服这一“不可成药"的难题,如疫苗,TCR-T疗法等。KRAS可以被细胞外信号以及来自细胞器等亚细胞结构的信号所激活。在正常的生理环境下,野生型KRAS主要处于不活跃的GDP结合状态。信号促使KRAS脱离GDP,并与GTP结合,将KRAS转化为激活状态。激活的KRAS能够结合并激活效应蛋白,如RAF激酶、PI3K和RalGDS,促进下游级联的信号通路,如MAPK、PI3K和Ral-GEFs通路。这些信号通路通过细胞复制、生长、分化和促进细胞周期进程。一个激活的致癌KRAS基因导致一个突变的KRAS蛋白,由于GTP水解率大大降低,异常长时间保持其活性GTP结合状态。当KRAS蛋白被困在“ON"位置时,下游效应通路也保持活跃,细胞增殖率保持很高。其他仍然活跃的效应通路包括那些促进肿瘤发展过程的通路,如有丝分裂、细胞迁移、肿瘤侵袭和血管生成。 一、高通量筛选试剂盒HTRF®技术提供均相,免洗KRAS结合检测试剂盒,通过这些已验证的试剂盒,可直接用于抑制剂筛选,无需优化,每个试剂盒配有重组蛋白、检测试剂和缓冲液。如下图所示,使用抗Tag1-SOS1(供体)和抗Tag2-XL665(受体)检测Tag1-SOS1与Tag2-KRAS-G12C之间的相互作用。当供体和受体抗体由于SOS1和KRAS-G12C结合而接近时,供体抗体的激发触发向受体抗体的荧光共振能量转移(FRET),受体抗体在665nm处特异性发射。这种特异性信号与KRAS-G12C/SOS1相互作用的程度成正比。因此,化合物或抗体阻断KRAS-G12C/SOS1的相互作用会导致HTRF®信号的减少。  
 使用已知抑制剂对试剂盒进行验证,均获得相应较好的IC50并且与已报道文献结果相一致。 
相关推荐:

货号产品名称规格
63ADK000CB15PEGKRAS-WT/SOS1 binding assay kit500 tests
63ADK000CB19PEGKRAS-WT/RAF1 binding assay kit500 tests
63ADK000CB47PEGKRAS-G12D/RAF1 binding assay kit500 tests
63ADK000CB17PEGKRAS-G12D/SOS1 binding assay kit500 tests
63ADK000CB20PEGKRAS-G12C/RAF1 binding assay kit500 tests
63ADK000CB16PEGKRAS-G12C/SOS1 binding assay kit500 tests
63ADK000CB22PEGKRAS-G12V/RAF1 binding assay kit500 tests
63ADK000CB28PEGKRAS G12V GTP binding kit500 tests

 二、KRAS靶点相关蛋白全部KRAS相关蛋白均已原核表达系统(E.coli)进行表达。通过HTRF方式验证其结合活性。 1、KRAS WTThe KRAS-WT activity was assayed with HTRF technology. The reaction was performed by incubating the KRAS- WT protein, GTP, cRAF and beads at 25℃ for 60 min, then reading Ratio 665/620nm signal.

 

2.KRAS-G12CThe KRAS-G12C activity was assayed with HTRF technology. The reaction was performed by incubating the KRAS-G12C protein, GTP, cRAF and beads at 25℃ for 60 min, then reading Ratio 665/620nm signal. 3.KRAS-G12D The KRAS-G12D activity was assayed with HTRF technology. The reaction was performed by incubating the KRAS-G12D protein, GTP, cRAF and beads at 25℃ for 60 min, then reading Ratio 665/620nm signal
4.KRAS-G12R
The KRAS-G12R activity was assayed with HTRF technology. The reaction was performed by incubating the KRAS-G12R protein, GTP, cRAF and beads at 25℃ for 60 min, then reading Ratio 665/620nm signal.

5.KRAS-G12VThe KRAS- G12V activity was assayed with HTRF technology. The reaction was performed by incubating the KRAS- G12V protein, GTP, cRAF and beads at 25℃ for 60 min, then reading Ratio 665/620nm signal.
6.KRAS-G13C
The KRAS-G13C activity was assayed with HTRF technology. The reaction was performed by incubating the KRAS-G13C protein, GTP, cRAF and beads at 25℃ for 60 min, then reading Ratio 665/620nm signal.
7.KRAS Q61H
The KRAS-Q61H activity was assayed with HTRF technology. The reaction was performed by incubating the KRAS-Q61H protein, GTP, cRAF and beads at 25℃ for 60 min, then reading Ratio 665/620nm signal.

8.SOS1 

9.RAF
相关推荐:

货号品名规格
UA080001KRAS100μg
UA080002Kras[G12C]100μg
UA080003Kras[G12V]100μg
UA080004Kras[G12D]100μg
UA080005Kras[G12R]100μg
UA080006Kras[G13C]100μg
UA080007Kras[G13D]100μg
UA080008Kras[Q61H]100μg
UA080014cRAF50μg
UA080015cRAF50μg
UA080016SOS1100μg

*以上KRAS蛋白无GTP bound,如需GTP bound,可联系优宁维。上述蛋白,常规规格有:50μg,100μg和1mg,如需更大规格,可联系优宁维定制。 三、Anti-Tag抗体话说,不想用试剂盒的情况下,如何建立assay呢?蛋白已经有了,还差检测抗体...这不就来了!Anti-Tag的HTRF荧光抗体,在实现高通量,操作简便等其他技术优势的同时,可以为assay的建立提供最大的灵活性。无关乎蛋白本身,仅针对标签如His,GST,FLAG等,选择对应的配对抗体即可实现实验方法学开发。比如:参考上述蛋白KRAS G12C(his Tag)和SOS1(GST tag),选择抗体配对有几种方式可供参考:

KRAS donorSOS1 accpetor
Anti his EuAnti GST XL665
Anti GST D2
Anti His TbAnti GST XL665
Anti GST D2
KRAS accpetorSOS1 donor
Anti his XL665Anti GST Eu
Anti his D2
Anti his XL665Anti GST Tb
Anti his D2
PPI - Europium detection buffer
PPI - Terbium detection buffer 

 下图为Anti-GST-Tb SOS1与Anti-his-XL665 KRAS G12C搭配组合展示。 
相关推荐:

  Anti-tag antibodies 
货号品名规格
61HISDLFMAb Anti 6HIS-d21000 tests
61HISKLAMAb Anti 6HIS-Eu cryptate5000 tests
61HI2KLAMAb Anti 6HIS-Eu cryptate Gold5000  tests
61HISTLFMAb Anti 6HIS-Tb cryptate1000 tests
61HI2TLFMAb Anti 6HIS-Tb cryptate Gold1000 tests
61HISXLFMAb Anti 6HIS-XL6651000 tests
61MYCDAFMAb Anti cmyc-d21000 tests
61MYCKLAMAb Anti cmyc-Eu cryptate5000 tests
61MYCTAFMAb Anti cmyc-Tb cryptate1000 tests
61MYCXLFMAb Anti cmyc-XL6651000 tests
61DNPKLAMAb Anti DNP-Eu cryptate5000 tests
61DNPXLAMAb Anti DNP-XL6655000 tests
61FG2DLFMAb Anti FLAG M2-d21000 tests
61FG2KLAMAb Anti FLAG M2-Eu cryptate5000 tests
61FG2TLFMAb Anti FLAG M2-Tb cryptate1000 tests
61FG2XLFMAb Anti FLAG M2-XL6651000 tests
61GSTDLFMAb Anti GST-d21000 tests
61GSTKLAMAb Anti GST-Eu cryptate5000 tests
61GSTTLFMAb Anti GST-Tb cryptate1000 tests
61GSTXLFMAb Anti GST-XL6651000 tests
610HADAFMAb Anti HA-d21000 tests
610HAKLAMAb Anti HA-Eu cryptate5000 tests
610HATAFMAb Anti HA-Tb cryptate1000 tests
610HAXLFMAb Anti HA-XL6651000 tests
61MBPDAAMAb Anti MBP-d25000 tests
61MBPKAAMAb Anti MBP-Eu cryptate5000 tests
61MBPTAAMAb Anti MBP-Tb cryptate5000 tests
Buffers
货号品名规格
61DB9RDF PPI - Europium detection buffer 200ml
61DB10RDF PPI - Terbium detection buffer 200ml

 三、下游磷酸化蛋白检测Alpha®和HTRF®技术基于细胞水平进行下游KRAS信号通路的研究,包括MAPK、PI3K和Ral-GEFs通路。这些细胞信号分析能够区分化合物调节下游KRAS通路的功效。HTRF®仍然利用夹心法原理,标记的特异性抗体识别磷酸化蛋白和总蛋白从而进行定量检测。全过程无需洗涤,特异性好,灵敏度高。 

下图为p-ERK(图左)和p-NFkB(图右)磷酸化试剂盒灵敏度与WB对比,灵敏度高出30倍不等。


与HTRF®技术不同,Alpha®虽然也是均相检测检测技术,但其基于化学发光原理,利用两个微珠进行检测来实现更低的实验背景和超高灵敏度。一个抗体被生物素化并与链霉亲和素包被的供体微珠结合,受体微珠则直接与另一个抗体结合。抗体与磷酸化蛋白(总蛋白)结合使供体和受体微珠靠近。供体微珠的激发使得单线态氧扩散至受体微珠,从而发射615nm波长的光信号。Alpha®的检测信号与样本中存在的磷酸化蛋白(总蛋白)的数量成正比。Alpha®原理如下图。
 

相关推荐:

货号产品名称规格
64NFBPEGNFkB phospho-S536 kit500tests
64NFTPEGNFkB total kit500tests
64AERPEGAdvanced phospho-ERK (Thr202/Tyr204)cellular kit500tests
64NRKPEGTotal ERK cellular kit500tests
64ME1PEGPhospho-MEK1 (Ser218/222) cellular kit500tests
64NE1PEGTotal MEK1 cellular kit500tests
64AKTPEGPhospho-AKT1/2/3 (Thr308) cellular kit500tests
64AKSPEGPhospho-AKT1/2/3 (Ser473) cellular kit500tests
64NKTPEGTotal AKT cellular kit500tests
64AKTPEGHTRF PHOSPHO-AKT (THR308) DETECTION KITS500tests
64TORPEGPhospho-mTOR (Ser2448) cellular kit500tests
ALSU-PAKT-B500AlphaLISA SureFire Ultra AKT 1/2/3 (pS473) Assay Kit500assays
ALSU-TAKT-B500Total AKT1/2/3 AlphaLISA SureFire Ultra Detection Kit500assays
ALSU-PMTOR-C500AlphaLISA phospho-mTOR (Ser2448) Assay Kit500assays
ALSU-TMTOR-B500AlphaLISA total mTOR Assay Kit500assays

 四、3D细胞增殖


基于ATP的发光法测定细胞增殖和细胞毒性的分析原理。用适当缓冲液直接裂解细胞后,对细胞ATP进行定量。在裂解细胞释放的ATP作用下,ATP与添加的荧光素反应产生光。

ATPlite 3D and ATPlite 1step 3D Assay 操作流程如下:


ATPlite 3D检测可以允许细胞密度较高情况下进行检测,并且比2D检测效果明显。

相关推荐:

货号产品名称规格
6066736ATPlite 1step 3D10ml/100tests
6066943ATPlite 3D300tests
6016736ATPlite 1step Luminescence Assay System10ml
6016731100ml
60167391000ml
6016943300 Assay
6016941ATPlite Luminescence Assay System1000 Assay
60169475000 Assay
601694910000 Assay


四、微孔板耗材
1.3D细胞培养板①板底为高度生物相容的,超低吸附力聚合物包被du有的合成聚合物MPC,源于细胞膜组分,与极性基团的磷脂(磷脂酰胆碱)具有相同的结构,提供自然生长环境,并消除批间差异。②易形成形态大小均匀的球体细胞球体高质量形成率,细胞形态均已一致,板材的均匀性高,可随时聚焦进行图片拍摄,提供重复性更高的数据及更高效更高质量的成像。③无需更换培养基实验的佳选择圆底板,性价比高。
 相关推荐:

货号产品名称规格
6055330CellCarrier Spheroid ULA MicroplatesCase of 10
6055334Case of 40


2. 其他相关培养板,检测板

底透微孔板(View Plate)
实验应用颜色特点孔数目Case of 40Case of 50Case of 60Case of 160
化学发光实验白色底透,TC处理96孔
6005181

384孔
6007480

黑色底透,TC处理96孔
6005182

384孔6007460


标准微孔板(OptiPlate)
实验应用颜色特点孔数目Case of 40Case of 50Case of 60Case of 160
化学发光实验荧光实验白色非底透96孔
6005290

384孔
6007290

黑色非底透,TC处理96孔
6005270

384孔
6007270

细胞培养板(CulturPlate)
实验应用颜色特点孔数目
Case of 50Case of 160Case of 200
细胞培养白色非底透,TC处理24孔
6005168

96孔
600568060056886005689
384孔
600768060076886007689
黑色非底透,TC处理96孔
600566060056686005669
384孔
60076606007668
             HTRF*Alpha检测微孔板
应用领域颜色特点孔数目Case of 25Case of 50
Case of 200
HTRF实验白色非底透,潜孔96孔66PL96025

6005359
Alpha实验384孔
6008280
6008289


更多实验相关微孔板,请联系优宁维。

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