KRAS(G12V) His Tag Protein, Human

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

Datasheet COA

价格￥1,490.00 （供应商现货： 3-5个工作日）

货号 UA085048

规格

10μg50μg100μg

数量

产品介绍

产品介绍评论(0)

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(G12V) 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 （G12V）His Tag Protein, Human was determined to be > 400 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

50mM Tris, 200mM NaCl, 20% Glycerol, 1mM DTT, pH7.5
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.Wang X, Wang W, Zou S, Xu Z, Cao D, Zhang S, Wei M, Zhan Q, Wen C, Li F, Chen H, Fu D, Jiang L, Zhao M, Shen B. Combination therapy of KRAS G12V mRNA vaccine and pembrolizumab: clinical benefit in patients with advanced solid tumors. Cell Res. 2024 Sep;34(9):661-664.
2.Li D, Geng K, Hao Y, Gu J, Kumar S, Olson AT, Kuismi CC, Kim HM, Pan Y, Sherman F, Williams AM, Li Y, Li F, Chen T, Thakurdin C, Ranieri M, Meynardie M, Levin DS, Stephens J, Chafitz A, Chen J, Donald-Paladino MS, Powell JM, Zhang ZY, Chen W, Ploszaj M, Han H, Gu SS, Zhang T, Hu B, Nacev BA, Kaiza ME, Berger AH, Wang X, Li J, Sun X, Liu Y, Zhang X, Bruno TC, Gray NS, Nabet B, Wong KK, Zhang H. Targeted degradation of oncogenic KRASG12V triggers antitumor immunity in lung cancer models. J Clin Invest. 2024 Dec 24;135(2):e174249.

背景介绍

KRAS (Kirsten rat sarcoma viral oncogene homolog) is a small GTPase that functions as a molecular switch regulating cell proliferation, differentiation, and survival signaling. Its protein structure consists of an N-terminal catalytic G domain (containing the P-loop, switch I/II regions, and the G12V mutation site) and a C-terminal hypervariable region (CAAX box). The G12V mutation locks the switch II region in a constitutively active GTP-bound state, abolishing GTPase activity and leading to persistent activation of downstream pathways such as RAF-MEK-ERK, PI3K-AKT, and RAL-GEF, thereby driving tumorigenesis. Clinically, KRAS G12V is one of the most common KRAS mutation subtypes in pancreatic cancer (approximately 30%), colorectal cancer, and lung cancer. Due to the lack of a cysteine residue for covalent targeting, it cannot be treated with G12C inhibitors, and therapeutic options have long been limited. In recent years, emerging strategies—including Pan-KRAS inhibitors, PROTAC degraders, mRNA vaccines targeting G12V neoantigens, and TCR-T cell therapies—have achieved breakthroughs, offering new precision treatment directions to overcome this "undruggable" target.

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(G12V) 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

生物活性
- The specific activity of KRAS （G12V）His Tag Protein, Human was determined to be > 400 pmol/min/mg in a GTPase-Glo assay using GTP solution substrate.
电泳
- 1μg (R: reducing condition, N:non-reducing condition).