圖1.

酯測試Western Blotting能夠適用于所有E2綴合酶。操作步驟按照“Assay Protocol”部分所述的進(jìn)行。

生物素化-泛素-酶共聚物通過蛋白質(zhì)印跡法的硫酯測試來檢測，其中分別包括：

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”部分所述的鏈霉親和素-辣根過氧化物酶檢測系統(tǒng)的有M: 生物素化SDS分子量標(biāo)記 (Sigma, SDS-6B) 從下到上: 20.1, 29.0, 39.8, 58.1 kDa。

應(yīng)用注釋

用法：

1. 在專用的E3泛素連接酶參與下對靶蛋白進(jìn)行泛素化。E2的家族能夠產(chǎn)生E2-Ub硫酯

    綴合物以用于測試，以及與特定E3/靶蛋白的結(jié)合。

    例如：p53在mdm2（E3）和UbcH5b（E2）10的參與下進(jìn)行泛素化。

2. 活化用于將硫酯與新型E2酶綴合的泛素。（在直接可比的條件下，可使用試劑盒中

    的E2s作為替代）

3. 使用細(xì)胞裂解液或粗提物/制備液作為E3泛素連接酶的來源，在泛素化試劑盒組分的

    參與下，促進(jìn)已純化的靶蛋白的泛素化。

4. 不依賴底物（目標(biāo)蛋白）的體外泛素化反應(yīng)。確定E3酶的泛素連接活性/特異性以及

    它們的催化結(jié)構(gòu)域/片段11。

注：應(yīng)用1和2提供的方案可以通過包含、省略或替代特定的酶組分來修改，從而應(yīng)用

       于其他實驗。

質(zhì)量

試劑盒包含了足夠進(jìn)行4次含有E2酶反應(yīng)的材料。

使用/穩(wěn)定性

試劑盒的所有組分應(yīng)在-80°C下儲存以確保穩(wěn)定性和活性。

儲存

避免反復(fù)凍融

運(yùn)輸

干冰運(yùn)輸

長期儲存

-80℃

組分

20×泛素活化酶溶液
人，重組 E1（His6-tagged）, 125 μL, 產(chǎn)品編號BML-UW9410
   
10× 泛素綴合酶溶液 （E2）
每種E2酶有20 μL
UbcH1（His6-tagged）,產(chǎn)品編號：BML-UW9020
UbcH2（His6-tagged）,產(chǎn)品編號：BML-UW9025
UbcH3（His6-tagged）,產(chǎn)品編號：BML-UW8730
UbcH5a（His6-tagged）,產(chǎn)品編號：BML-UW9050
UbcH5b（His6-tagged）,產(chǎn)品編號：BML-UW9060
UbcH5c（His6-tagged）,產(chǎn)品編號：BML-UW9070
UbcH6（His6-tagged）,產(chǎn)品編號：BML-UW8710
UbcH7（His6-tagged）,產(chǎn)品編號：BML-UW9080
UbcH8（His6-tagged）,產(chǎn)品編號：BML-UW9135
UbcH10（untagged）,產(chǎn)品編號：BML-UW0960
UbcH13/Mms2（His6-tagged）,產(chǎn)品編號：BML-UW9565
   
20×生物素化泛素溶液 （Bt-Ub）, 125 μL,產(chǎn)品編號：BML-UW8705
20× Mg-ATP溶液, 125 μL,產(chǎn)品編號：BML-EW9805
2×非還原性凝膠上樣緩沖液, 2.5 mL,產(chǎn)品編號：BML-KW9880
10× 泛素化緩沖液, 250 μL,產(chǎn)品編號：BML-KW9885

產(chǎn)品編號

產(chǎn)品名稱

產(chǎn)品規(guī)格

BML-UW9920-0001

Ubiquitinylation kit
泛素化試劑盒

1 kit

產(chǎn)品編號

產(chǎn)品名稱

產(chǎn)品規(guī)格

BML-UW8995-0001

UBI-QAPTURE-Q kit
純化試劑

20 tests

BML-UW9915-0001

Ubiquitin   conjugating kit (HeLa lysate-based)
泛素結(jié)合試劑盒（HeLa細(xì)胞裂解液）   

20 tests

BML-UW0970-0001

Auto-ubiquitinylation kit
自泛素化試劑盒

10 tests

BML-UW0590-0001

NEDDylation Kit
NEDD化試劑盒

20 tests

BML-UW0400A-0001

Ubiquitin activating kit
泛素激活試劑盒

96 wells

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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

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10.

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

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(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;

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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;

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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;