图1.

酯测试Western Blotting能够适用于所有E2缀合酶。操作步骤按照“Assay Protocol”部分所述的进行。

生物素化-泛素-酶共聚物通过蛋白质印迹法的硫酯测试来检测，其中分别包括：

A: UbcH1 (Prod. No. BML-UW9020)、B: UbcH2 (Prod. No. BML-UW9025)、

C: UbcH3 (Prod. No. BML-UW8730)、D: UbcH5a (Prod. No. BML-UW9050)、

E: UbcH5b (Prod. No. BML-UW9060)、F: UbcH5c (Prod. No. BML-UW9070)、

G: UbcH6 (Prod. No. BML-UW8710)、H: UbcH7 (Prod. No. BML-UW9080)、

I: UbcH8 (Prod. No. BML-UW9135)、J: UbcH10 (Prod. No. BML-UW0960)、

K: Ubc13/MMS2 (Prod. No. BML-UW9565)，

而使用“Analysis by Western Blotting”部分所述的链霉亲和素-辣根过氧化物酶检测系统的有M: 生物素化SDS分子量标记 (Sigma, SDS-6B) 从下到上: 20.1, 29.0, 39.8, 58.1 kDa。

应用注释

用法：

1. 在专用的E3泛素连接酶参与下对靶蛋白进行泛素化。E2的家族能够产生E2-Ub硫酯

    缀合物以用于测试，以及与特定E3/靶蛋白的结合。

    例如：p53在mdm2（E3）和UbcH5b（E2）10的参与下进行泛素化。

2. 活化用于将硫酯与新型E2酶缀合的泛素。（在直接可比的条件下，可使用试剂盒中

    的E2s作为替代）

3. 使用细胞裂解液或粗提物/制备液作为E3泛素连接酶的来源，在泛素化试剂盒组分的

    参与下，促进已纯化的靶蛋白的泛素化。

4. 不依赖底物（目标蛋白）的体外泛素化反应。确定E3酶的泛素连接活性/特异性以及

    它们的催化结构域/片段11。

注：应用1和2提供的方案可以通过包含、省略或替代特定的酶组分来修改，从而应用

       于其他实验。

质量

试剂盒包含了足够进行4次含有E2酶反应的材料。

使用/稳定性

试剂盒的所有组分应在-80°C下储存以确保稳定性和活性。

储存

避免反复冻融

运输

干冰运输

长期储存

-80℃

组分

20×泛素活化酶溶液
人，重组 E1（His6-tagged）, 125 µL, 产品编号BML-UW9410
   
10× 泛素缀合酶溶液 （E2）
每种E2酶有20 µL
UbcH1（His6-tagged）,产品编号：BML-UW9020
UbcH2（His6-tagged）,产品编号：BML-UW9025
UbcH3（His6-tagged）,产品编号：BML-UW8730
UbcH5a（His6-tagged）,产品编号：BML-UW9050
UbcH5b（His6-tagged）,产品编号：BML-UW9060
UbcH5c（His6-tagged）,产品编号：BML-UW9070
UbcH6（His6-tagged）,产品编号：BML-UW8710
UbcH7（His6-tagged）,产品编号：BML-UW9080
UbcH8（His6-tagged）,产品编号：BML-UW9135
UbcH10（untagged）,产品编号：BML-UW0960
UbcH13/Mms2（His6-tagged）,产品编号：BML-UW9565
   
20×生物素化泛素溶液 （Bt-Ub）, 125 µL,产品编号：BML-UW8705
20× Mg-ATP溶液, 125 µL,产品编号：BML-EW9805
2×非还原性凝胶上样缓冲液, 2.5 mL,产品编号：BML-KW9880
10× 泛素化缓冲液, 250 µL,产品编号：BML-KW9885

产品编号

产品名称

产品规格

BML-UW9920-0001

Ubiquitinylation kit
泛素化试剂盒

1 kit

产品编号

产品名称

产品规格

BML-UW8995-0001

UBI-QAPTURE-Q kit
纯化试剂

20 tests

BML-UW9915-0001

Ubiquitin   conjugating kit (HeLa lysate-based)
泛素结合试剂盒（HeLa细胞裂解液）   

20 tests

BML-UW0970-0001

Auto-ubiquitinylation kit
自泛素化试剂盒

10 tests

BML-UW0590-0001

NEDDylation Kit
NEDD化试剂盒

20 tests

BML-UW0400A-0001

Ubiquitin activating kit
泛素激活试剂盒

96 wells

  1.

BTK blocks the inhibitory effects of MDM2 on p53 activity: M. Rada, et al.; Oncotarget 8, 106639 (2017), Abstract; Full Text

  2.

Hepatocyte TRAF3 promotes liver steatosis and systemic insulin resistance through targeting TAK1-dependent signalling: P.X. Wang, et al.; Nat. Commun. 7, 10592 (2016), Application(s):

In vitro ubiquitination assays, Abstract; Full Text

  3.

Host cell-catalyzed S-palmitoylation mediates Golgi targeting of the Legionella ubiquitin

ligase GobX: Y.H. Lin, et al.; J. Biol. Chem. 290, 25766 (2015), Application(s): Western Blot,

Abstract; Full Text

  4.

TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING: Y. Wang, et al.; PLoS Pathog. 11, e1005012 (2015),

 Application(s): In vitro ubiquitination assay, Abstract; Full Text

  5.

TRIM35 negatively regulates TLR7- and TLR9-mediated type I interferon production by targeting IRF7: Y. Wang, et al.; FEBS Lett. 589, 1322 (2015), Application(s): In vitro

ubiquitination assay, Abstract;

  6.

Parkin loss of function contributes to RTP801 elevation and neurodegeneration in

Parkinson's disease: J. Romaní-Aumedes, et al.; Cell Death Dis. 5, e1364 (2014), Abstract;

  7.

Rlim, an E3 ubiquitin ligase, influences the stability of Stathmin protein in human

osteosarcoma cells: X. Chen, et al.; Cell Signal. 26, 1532 (2014), Abstract;

  8.

Stability of the human pregnane X receptor is regulated by E3 ligase UBR5 and

serine/threonine kinase DYRK2: S.S. Ong, et al.; Biochem. J. 459, 193 (2014), Abstract;

  9.

Ubiquitin-proteasome-mediated degradation of S-RNase in a solanaceous

cross-compatibility reaction: T. Entani, et al.; Plant J. 78, 1014 (2014), Abstract;

10.

E3 ubiquitin ligase RNF126 promotes cancer cell proliferation by targeting the tumor

suppressor p21 for ubiquitin-mediated degradation: X. Zhi, et al.; Cancer Res. 73, 385

(2013), Abstract;

11.

The E3 ubiquitin ligase MARCH8 negatively regulates IL-1β-induced NF-κB activation by

targeting the IL1RAP coreceptor for ubiquitination and degradation: R. Chen, et al.; Proc.

Natl. Acad. Sci. U.S.A. 109, 14128 (2012), Abstract; Full Text

12.

TRIM32 protein modulates type I interferon induction and cellular antiviral response by targeting MITA/STING protein for K63-linked ubiquitination: J. Zhang, et al.; J. Biol. Chem. 287, 28646 (2012), Abstract;

13.

Cullin 4B protein ubiquitin ligase targets peroxiredoxin III for degradation: X. Li, et al.; J.

Biol. Chem. 286, 32344 (2011), Abstract; Full Text

14.

Tripartite motif 8 (TRIM8) modulates TNFα- and IL-1β-triggered NF-κB activation by

targeting TAK1 for K63-linked polyubiquitination: Q. Li, et al.; Proc. Natl. Acad. Sci. U.S.A. 

108, 19341 (2011), Abstract; Full Text

15.

1-Benzyl-1,2,3,4-tetrahydroisoquinoline binds with tubulin beta, a substrate of parkin, and

reduces its polyubiquitination: R. Kohta, et al.; J. Neurochem. 114, 1291 (2010), Abstract;

16.

Pasteurella multocida Toxin-induced Pim-1 expression disrupts suppressor of cytokine

signalling (SOCS)-1 activity: D. Hildebrand, et al.; Cell. Microbiol. 12, 1732 (2010), Abstract;

17.

The E3 ubiquitin ligase RNF5 targets virus-induced signaling adaptor for ubiquitination and

degradation: B. Zhong, et al.; J. Immunol. 184, 6249 (2010), Abstract; Full Text

18.

The Fbw7 tumor suppressor targets KLF5 for ubiquitin-mediated degradation and

suppresses breast cell proliferation: D. Zhao, et al.; Cancer Res. 70, 4728 (2010), 

Abstract; Full Text

19.

REUL is a novel E3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-I: D. Gao, et al.; PLoS One 4, e5760 (2009), Abstract; Full Text

  1.

Regulation of p53 by the ubiquitin-conjugating enzymes UbcH5B/C in vivo: M.K. Saville, et al.;

J. Biol. Chem. 279, 42169 (42169), Abstract;

  2.

Characterization of the novel E3 ubiquitin ligase encoded in exon 3 of herpes simplex

virus-1-infected cell protein 0: R. Hagglund, et al.; PNAS 99, 7889 (2002), Abstract;

  3.

Mechanisms underlying ubiquitination: C.M. Pickart, et al.; Annu. Rev. Biochem. 70, 503 (2001),

Abstract;

  4.

Functions of the MDM2 oncoprotein: D.A. Freedman; Cell Mol. Life. Sci. 55, 96 (1999), Abstract;

  5.

Regulation of the p53 tumor suppressor protein: M. Oren, et al.; J. Biol. Chem. 274, 36031

(1999), Abstract;

  6.

The p53 pathway: C. Prives, et al.; J. Pathol. 187, 112 (1999), Abstract;

  7.

The ubiquitin-proteasome system and endocytosis: G.J. Strous, et al.; J. Cell. Sci. 112 ,

1417 (1999), Abstract;

  8.

The ubiquitin system: A. Hershko, et al.; Annu. Rev. Biochem. 67, 425 (1998), Abstract;

  9.

Pathways of ubiquitin conjugation: A.L. Haas, et al.; FASEB J. 11, 1257 (1997), Abstract;

10.

P53 and mdm-2: interactions between tumor suppressor gene and oncogene products:

M. Perry, et al.; Mt. Sinai J. Med. 61, 291 (1994), Abstract;

11.

The p53 tumor suppressor gene: A.J. Levine, et al.; J. Lab. Clin. Med. 123, 817 (1994), Abstract;