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KRAS靶點(diǎn)難成藥？-整體解決方案來幫您
KRAS熱度已經(jīng)消退了？NO...據(jù)調(diào)查，現(xiàn)在除傳統(tǒng)針對(duì)KRAS靶點(diǎn)小分子藥物外，更多玩家嘗試新興療法征服這一“不可成藥"的難題，如疫苗，TCR-T療法等。KRAS可以被細(xì)胞外信號(hào)以及來自細(xì)胞器等亞細(xì)胞結(jié)構(gòu)的信號(hào)所激活。在正常的生理環(huán)境下，野生型KRAS主要處于不活躍的GDP結(jié)合狀態(tài)。信號(hào)促使KRAS脫離GDP，并與GTP結(jié)合，將KRAS轉(zhuǎn)化為激活狀態(tài)。激活的KRAS能夠結(jié)合并激活效應(yīng)蛋白，如RAF激酶、PI3K和RalGDS，促進(jìn)下游級(jí)聯(lián)的信號(hào)通路，如MAPK、PI3K和Ral-GEFs通路。這些信號(hào)通路通過細(xì)胞復(fù)制、生長(zhǎng)、分化和促進(jìn)細(xì)胞周期進(jìn)程。一個(gè)激活的致癌KRAS基因?qū)е乱粋€(gè)突變的KRAS蛋白，由于GTP水解率大大降低，異常長(zhǎng)時(shí)間保持其活性GTP結(jié)合狀態(tài)。當(dāng)KRAS蛋白被困在“ON"位置時(shí)，下游效應(yīng)通路也保持活躍，細(xì)胞增殖率保持很高。其他仍然活躍的效應(yīng)通路包括那些促進(jìn)腫瘤發(fā)展過程的通路，如有絲分裂、細(xì)胞遷移、腫瘤侵襲和血管生成。 一、高通量篩選試劑盒HTRF®技術(shù)提供均相，免洗KRAS結(jié)合檢測(cè)試劑盒，通過這些已驗(yàn)證的試劑盒，可直接用于抑制劑篩選，無需優(yōu)化，每個(gè)試劑盒配有重組蛋白、檢測(cè)試劑和緩沖液。如下圖所示，使用抗Tag1-SOS1（供體）和抗Tag2-XL665（受體）檢測(cè)Tag1-SOS1與Tag2-KRAS-G12C之間的相互作用。當(dāng)供體和受體抗體由于SOS1和KRAS-G12C結(jié)合而接近時(shí)，供體抗體的激發(fā)觸發(fā)向受體抗體的熒光共振能量轉(zhuǎn)移（FRET），受體抗體在665nm處特異性發(fā)射。這種特異性信號(hào)與KRAS-G12C/SOS1相互作用的程度成正比。因此，化合物或抗體阻斷KRAS-G12C/SOS1的相互作用會(huì)導(dǎo)致HTRF®信號(hào)的減少。 
使用已知抑制劑對(duì)試劑盒進(jìn)行驗(yàn)證，均獲得相應(yīng)較好的IC50并且與已報(bào)道文獻(xiàn)結(jié)果相一致。 
相關(guān)推薦：
| 貨號(hào) | 產(chǎn)品名稱 | 規(guī)格 |
| 63ADK000CB15PEG | KRAS-WT/SOS1 binding assay kit | 500 tests |
| 63ADK000CB19PEG | KRAS-WT/RAF1 binding assay kit | 500 tests |
| 63ADK000CB47PEG | KRAS-G12D/RAF1 binding assay kit | 500 tests |
| 63ADK000CB17PEG | KRAS-G12D/SOS1 binding assay kit | 500 tests |
| 63ADK000CB20PEG | KRAS-G12C/RAF1 binding assay kit | 500 tests |
| 63ADK000CB16PEG | KRAS-G12C/SOS1 binding assay kit | 500 tests |
| 63ADK000CB22PEG | KRAS-G12V/RAF1 binding assay kit | 500 tests |
| 63ADK000CB28PEG | KRAS G12V GTP binding kit | 500 tests |
二、KRAS靶點(diǎn)相關(guān)蛋白全部KRAS相關(guān)蛋白均已原核表達(dá)系統(tǒng)（E.coli）進(jìn)行表達(dá)。通過HTRF方式驗(yàn)證其結(jié)合活性。 1、KRAS WT
The 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-G12C
The 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-G12V
The 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
相關(guān)推薦：
| 貨號(hào) | 品名 | 規(guī)格 |
| UA080001 | KRAS | 100μg |
| UA080002 | Kras[G12C] | 100μg |
| UA080003 | Kras[G12V] | 100μg |
| UA080004 | Kras[G12D] | 100μg |
| UA080005 | Kras[G12R] | 100μg |
| UA080006 | Kras[G13C] | 100μg |
| UA080007 | Kras[G13D] | 100μg |
| UA080008 | Kras[Q61H] | 100μg |
| UA080014 | cRAF | 50μg |
| UA080015 | cRAF | 50μg |
| UA080016 | SOS1 | 100μg |
*以上KRAS蛋白無GTP bound，如需GTP bound，可聯(lián)系優(yōu)寧維。上述蛋白，常規(guī)規(guī)格有：50μg，100μg和1mg，如需更大規(guī)格，可聯(lián)系優(yōu)寧維定制。 三、Anti-Tag抗體話說，不想用試劑盒的情況下，如何建立assay呢？蛋白已經(jīng)有了，還差檢測(cè)抗體...這不就來了！Anti-Tag的HTRF熒光抗體，在實(shí)現(xiàn)高通量，操作簡(jiǎn)便等其他技術(shù)優(yōu)勢(shì)的同時(shí)，可以為assay的建立提供最大的靈活性。無關(guān)乎蛋白本身，僅針對(duì)標(biāo)簽如His，GST，F(xiàn)LAG等，選擇對(duì)應(yīng)的配對(duì)抗體即可實(shí)現(xiàn)實(shí)驗(yàn)方法學(xué)開發(fā)。比如：參考上述蛋白KRAS G12C（his Tag）和SOS1（GST tag），選擇抗體配對(duì)有幾種方式可供參考：
| KRAS donor | SOS1 accpetor |
| Anti his Eu | Anti GST XL665 |
| Anti GST D2 | |
| Anti His Tb | Anti GST XL665 |
| Anti GST D2 | |
| KRAS accpetor | SOS1 donor |
| Anti his XL665 | Anti GST Eu |
| Anti his D2 | |
| Anti his XL665 | Anti GST Tb |
| Anti his D2 | |
| PPI - Europium detection buffer | |
| PPI - Terbium detection buffer | |
下圖為Anti-GST-Tb SOS1與Anti-his-XL665 KRAS G12C搭配組合展示。 
相關(guān)推薦：
| Anti-tag antibodies | ||
| 貨號(hào) | 品名 | 規(guī)格 |
| 61HISDLF | MAb Anti 6HIS-d2 | 1000 tests |
| 61HISKLA | MAb Anti 6HIS-Eu cryptate | 5000 tests |
| 61HI2KLA | MAb Anti 6HIS-Eu cryptate Gold | 5000 tests |
| 61HISTLF | MAb Anti 6HIS-Tb cryptate | 1000 tests |
| 61HI2TLF | MAb Anti 6HIS-Tb cryptate Gold | 1000 tests |
| 61HISXLF | MAb Anti 6HIS-XL665 | 1000 tests |
| 61MYCDAF | MAb Anti cmyc-d2 | 1000 tests |
| 61MYCKLA | MAb Anti cmyc-Eu cryptate | 5000 tests |
| 61MYCTAF | MAb Anti cmyc-Tb cryptate | 1000 tests |
| 61MYCXLF | MAb Anti cmyc-XL665 | 1000 tests |
| 61DNPKLA | MAb Anti DNP-Eu cryptate | 5000 tests |
| 61DNPXLA | MAb Anti DNP-XL665 | 5000 tests |
| 61FG2DLF | MAb Anti FLAG M2-d2 | 1000 tests |
| 61FG2KLA | MAb Anti FLAG M2-Eu cryptate | 5000 tests |
| 61FG2TLF | MAb Anti FLAG M2-Tb cryptate | 1000 tests |
| 61FG2XLF | MAb Anti FLAG M2-XL665 | 1000 tests |
| 61GSTDLF | MAb Anti GST-d2 | 1000 tests |
| 61GSTKLA | MAb Anti GST-Eu cryptate | 5000 tests |
| 61GSTTLF | MAb Anti GST-Tb cryptate | 1000 tests |
| 61GSTXLF | MAb Anti GST-XL665 | 1000 tests |
| 610HADAF | MAb Anti HA-d2 | 1000 tests |
| 610HAKLA | MAb Anti HA-Eu cryptate | 5000 tests |
| 610HATAF | MAb Anti HA-Tb cryptate | 1000 tests |
| 610HAXLF | MAb Anti HA-XL665 | 1000 tests |
| 61MBPDAA | MAb Anti MBP-d2 | 5000 tests |
| 61MBPKAA | MAb Anti MBP-Eu cryptate | 5000 tests |
| 61MBPTAA | MAb Anti MBP-Tb cryptate | 5000 tests |
| Buffers | ||
| 貨號(hào) | 品名 | 規(guī)格 |
| 61DB9RDF | PPI - Europium detection buffer | 200ml |
| 61DB10RDF | PPI - Terbium detection buffer | 200ml |
三、下游磷酸化蛋白檢測(cè)Alpha®和HTRF®技術(shù)基于細(xì)胞水平進(jìn)行下游KRAS信號(hào)通路的研究，包括MAPK、PI3K和Ral-GEFs通路。這些細(xì)胞信號(hào)分析能夠區(qū)分化合物調(diào)節(jié)下游KRAS通路的功效。HTRF®仍然利用夾心法原理，標(biāo)記的特異性抗體識(shí)別磷酸化蛋白和總蛋白從而進(jìn)行定量檢測(cè)。全過程無需洗滌，特異性好，靈敏度高。 
下圖為p-ERK（圖左）和p-NFkB（圖右）磷酸化試劑盒靈敏度與WB對(duì)比，靈敏度高出30倍不等。
與HTRF®技術(shù)不同，Alpha®雖然也是均相檢測(cè)檢測(cè)技術(shù)，但其基于化學(xué)發(fā)光原理，利用兩個(gè)微珠進(jìn)行檢測(cè)來實(shí)現(xiàn)更低的實(shí)驗(yàn)背景和超高靈敏度。一個(gè)抗體被生物素化并與鏈霉親和素包被的供體微珠結(jié)合，受體微珠則直接與另一個(gè)抗體結(jié)合。抗體與磷酸化蛋白（總蛋白）結(jié)合使供體和受體微珠靠近。供體微珠的激發(fā)使得單線態(tài)氧擴(kuò)散至受體微珠，從而發(fā)射615nm波長(zhǎng)的光信號(hào)。Alpha®的檢測(cè)信號(hào)與樣本中存在的磷酸化蛋白（總蛋白）的數(shù)量成正比。Alpha®原理如下圖。 
相關(guān)推薦：
| 貨號(hào) | 產(chǎn)品名稱 | 規(guī)格 |
| 64NFBPEG | NFkB phospho-S536 kit | 500tests |
| 64NFTPEG | NFkB total kit | 500tests |
| 64AERPEG | Advanced phospho-ERK (Thr202/Tyr204)cellular kit | 500tests |
| 64NRKPEG | Total ERK cellular kit | 500tests |
| 64ME1PEG | Phospho-MEK1 (Ser218/222) cellular kit | 500tests |
| 64NE1PEG | Total MEK1 cellular kit | 500tests |
| 64AKTPEG | Phospho-AKT1/2/3 (Thr308) cellular kit | 500tests |
| 64AKSPEG | Phospho-AKT1/2/3 (Ser473) cellular kit | 500tests |
| 64NKTPEG | Total AKT cellular kit | 500tests |
| 64AKTPEG | HTRF PHOSPHO-AKT (THR308) DETECTION KITS | 500tests |
| 64TORPEG | Phospho-mTOR (Ser2448) cellular kit | 500tests |
| ALSU-PAKT-B500 | AlphaLISA SureFire Ultra AKT 1/2/3 (pS473) Assay Kit | 500assays |
| ALSU-TAKT-B500 | Total AKT1/2/3 AlphaLISA SureFire Ultra Detection Kit | 500assays |
| ALSU-PMTOR-C500 | AlphaLISA phospho-mTOR (Ser2448) Assay Kit | 500assays |
| ALSU-TMTOR-B500 | AlphaLISA total mTOR Assay Kit | 500assays |
四、3D細(xì)胞增殖
基于ATP的發(fā)光法測(cè)定細(xì)胞增殖和細(xì)胞毒性的分析原理。用適當(dāng)緩沖液直接裂解細(xì)胞后，對(duì)細(xì)胞ATP進(jìn)行定量。在裂解細(xì)胞釋放的ATP作用下，ATP與添加的熒光素反應(yīng)產(chǎn)生光。
ATPlite 3D and ATPlite 1step 3D Assay 操作流程如下：
ATPlite 3D檢測(cè)可以允許細(xì)胞密度較高情況下進(jìn)行檢測(cè)，并且比2D檢測(cè)效果明顯。
相關(guān)推薦：
| 貨號(hào) | 產(chǎn)品名稱 | 規(guī)格 |
| 6066736 | ATPlite 1step 3D | 10ml/100tests |
| 6066943 | ATPlite 3D | 300tests |
| 6016736 | ATPlite 1step Luminescence Assay System | 10ml |
| 6016731 | 100ml | |
| 6016739 | 1000ml | |
| 6016943 | 300 Assay | |
| 6016941 | ATPlite Luminescence Assay System | 1000 Assay |
| 6016947 | 5000 Assay | |
| 6016949 | 10000 Assay |
四、微孔板耗材1.3D細(xì)胞培養(yǎng)板①板底為高度生物相容的，超低吸附力聚合物包被du有的合成聚合物MPC，源于細(xì)胞膜組分，與極性基團(tuán)的磷脂（磷脂酰膽堿）具有相同的結(jié)構(gòu)，提供自然生長(zhǎng)環(huán)境，并消除批間差異。②易形成形態(tài)大小均勻的球體細(xì)胞球體高質(zhì)量形成率，細(xì)胞形態(tài)均已一致，板材的均勻性高，可隨時(shí)聚焦進(jìn)行圖片拍攝，提供重復(fù)性更高的數(shù)據(jù)及更高效更高質(zhì)量的成像。③無需更換培養(yǎng)基實(shí)驗(yàn)的佳選擇圓底板，性價(jià)比高。
相關(guān)推薦：
| 貨號(hào) | 產(chǎn)品名稱 | 規(guī)格 |
| 6055330 | CellCarrier Spheroid ULA Microplates | Case of 10 |
| 6055334 | Case of 40 |
2. 其他相關(guān)培養(yǎng)板，檢測(cè)板
| 底透微孔板（View Plate） | |||||||
| 實(shí)驗(yàn)應(yīng)用 | 顏色 | 特點(diǎn) | 孔數(shù)目 | Case of 40 | Case of 50 | Case of 60 | Case of 160 |
| 化學(xué)發(fā)光實(shí)驗(yàn) | 白色 | 底透，TC處理 | 96孔 | 6005181 | |||
| 384孔 | 6007480 | ||||||
| 黑色 | 底透，TC處理 | 96孔 | 6005182 | ||||
| 384孔 | 6007460 | ||||||
| 標(biāo)準(zhǔn)微孔板（OptiPlate） | |||||||
| 實(shí)驗(yàn)應(yīng)用 | 顏色 | 特點(diǎn) | 孔數(shù)目 | Case of 40 | Case of 50 | Case of 60 | Case of 160 |
| 化學(xué)發(fā)光實(shí)驗(yàn)熒光實(shí)驗(yàn) | 白色 | 非底透 | 96孔 | 6005290 | |||
| 384孔 | 6007290 | ||||||
| 黑色 | 非底透，TC處理 | 96孔 | 6005270 | ||||
| 384孔 | 6007270 | ||||||
| 細(xì)胞培養(yǎng)板（CulturPlate） | |||||||
| 實(shí)驗(yàn)應(yīng)用 | 顏色 | 特點(diǎn) | 孔數(shù)目 | Case of 50 | Case of 160 | Case of 200 | |
| 細(xì)胞培養(yǎng) | 白色 | 非底透，TC處理 | 24孔 | 6005168 | |||
| 96孔 | 6005680 | 6005688 | 6005689 | ||||
| 384孔 | 6007680 | 6007688 | 6007689 | ||||
| 黑色 | 非底透，TC處理 | 96孔 | 6005660 | 6005668 | 6005669 | ||
| 384孔 | 6007660 | 6007668 | |||||
| HTRF*Alpha檢測(cè)微孔板 | |||||||
| 應(yīng)用領(lǐng)域 | 顏色 | 特點(diǎn) | 孔數(shù)目 | Case of 25 | Case of 50 | Case of 200 | |
| HTRF實(shí)驗(yàn) | 白色 | 非底透，潛孔 | 96孔 | 66PL96025 | 6005359 | ||
| Alpha實(shí)驗(yàn) | 384孔 | 6008280 | 6008289 | ||||
更多實(shí)驗(yàn)相關(guān)微孔板，請(qǐng)聯(lián)系優(yōu)寧維。
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