KRAS(G12D) His Tag Protein, Human

K-Ras 2,Ki-Ras,c-K-ras,c-Ki-ras,GTPase KRas,KRAS2,RASK2

Datasheet COA

价格￥1,160.00 （供应商现货： 3-5个工作日）

货号 UA085040

规格

10μg50μg100μg

数量

产品介绍

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产品规格

物种

Human
分子别名

K-Ras 2, Ki-Ras, c-K-ras, c-Ki-ras, GTPase KRas, KRAS2, RASK2
Accession

P01116-2
表达序列

Thr2-Cys185（G12D）with His Tag at the C-Terminus
表达宿主

E.coli
分子量

20-25kDa (Reducing)
纯度

＞95% by SDS-PAGE
活性

The specific activity of KRAS（G12D）His Tag Protein, Human was determined to be > 250 pmol/min/mg in a GTPase-Glo assay using GTP solution substrate.
标记

Unconjugated
标签

His Tag
性状

Liquid
缓冲体系

20 mM Tris, 150 mM NaCl, 1 mM DTT, PH7.4 ,10% glycerol
溶解方法

Reconstitute at 0.1-1 mg/ml according to the size in ultrapure water after rapid centrifugation.
储存条件

Stable for 12 months upon stored at -80℃ from the date of receipt. And avoid repeated freeze-thaws cycles.
文献引用

1. Gremer L, Merbitz-Zahradnik T, Dvorsky R, Cirstea IC, Kratz CP, Zenker M, Wittinghofer A, Ahmadian MR. Germline KRAS mutations cause aberrant biochemical and physical properties leading to developmental disorders. Hum Mutat. 2011 Jan;32(1):33-43.
2. Serra RW, Fang M, Park SM, Hutchinson L, Green MR. A KRAS-directed transcriptional silencing pathway that mediates the CpG island methylator phenotype. Elife. 2014 Mar 12;3:e02313.
3. Sun Q, Burke JP, Phan J, Burns MC, Olejniczak ET, Waterson AG, Lee T, Rossanese OW, Fesik SW. Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angew Chem Int Ed Engl. 2012 Jun 18;51(25):6140-3.

背景介绍

The KRAS protein (Kirsten rat sarcoma viral oncogene homolog) is a small GTPase protein whose structure comprises a G-domain (responsible for GTP/GDP binding) and a hypervariable region. The G12D mutation refers to the substitution of glycine at position 12 (Gly12) with aspartic acid (Asp), which prevents GAP (GTPase-activating protein) from promoting GTP hydrolysis, thereby locking KRAS in a persistently GTP-bound "active" state. This leads to constitutive activation of downstream signaling pathways including RAF-MEK-ERK and PI3K-AKT, driving cellular proliferation, survival, and metabolic reprogramming. Clinically, KRAS G12D represents one of the most prevalent oncogenic mutations in pancreatic cancer (~40%), colorectal cancer, and lung cancer, and has historically been considered "undruggable." However, recent breakthroughs include the development of non-covalent inhibitors (binding the Switch II pocket via salt bridge formation), elucidation of synergistic tumorigenic mechanisms between PTEN loss and G12D, demonstration of G12D-induced immunosuppressive microenvironment conferring resistance to PD-1/PD-L1 inhibitors, and discovery of G12D-specific stem cell reprogramming in lung adenocarcinoma, offering novel directions for precision-targeted therapies and combination immunotherapeutic strategies.

操作步骤

实验方法
实验原理：GTPase Glo™ 检测法通过检测KRAS反应中GTP水解后剩余的GTP量来评估KRAS活性。
实验材料
1.KRAS(G12D) His Tag Protein, Human
2.GTPase Glo™ Assay (Promega, Catalog # V7681T)
3.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)
4.Plate Reader (PerkinElmer)
实验步骤
1.使用GTPase/GAP Buffer配制包含10 µM GTP和2 mM DTT 的2X GTP溶液。
2.将KRAS用GTPase/GAP Buffer稀释至100 µg/mL、80 µg/mL和60 µg/mL，然后分别向384孔板的每个孔中加入5 µL。
3.向每个孔中加入5 µL步骤1中配制的2X GTP溶液以启动反应。使用5 µL GTPase/GAP Buffer与2X GTP溶液作为空白对照。反应总体积为10 µL。
4.室温（22-25℃）下孵育反应30分钟。
5.轻轻颠倒混匀解冻后的GTPase Glo™ Reagent, 500X，请勿涡旋振荡。根据需要，按比例增减下述成分的体积，配制所需体积的复溶GTPase Glo™。
Sample Name Amount
GTPase Glo ™ Reagent, 500X 2 μL
ADP, 10 mM 0.5 μL
GTPase Glo ™ Buffer 998 μL
Total volume 1 mL

6.向完成反应的反应体系中加入10 µL复溶后的GTPase-Glo™ Reagent，短暂混匀后，在室温（22–25°C）下震荡孵育30分钟。
7.加入20 µL检测试剂，将酶标板在室温（22–25°C）下孵育5–10分钟。
8.分别在发射波长555 nm（luminescence）下以终点模式读数。
9.计算比活性。
Specific Activity (pmol/min/μg) = ( 1-Sample OD / BLANK OD)*50pmol
Incubation time(min)×amount of enzyme (μg)

样品OD值：剩余ATP的OD值
空白OD值：添加GTP的OD值
50 pmol：添加的GTP量
孵育时间：30分钟
酶量：0.5 μg、0.4 μg和0.3 μg