Recombinant Human FLAG (DYKDDDDK)-Ubiquitin Protein, CF Summary
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins.Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration.The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard.In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
U-120
| Formulation | Lyophilized from a solution in deionized water. |
| Reconstitution | Reconstitute at 10 mg/mL in an aqueous solution. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Reconstitution Calculator
Background: Ubiquitin
Ubiquitin is a 76 amino acid (aa) protein that is ubiquitously expressed in all eukaryotic organisms. Ubiquitin is highly conserved with 96% aa sequence identity shared between human and yeast Ubiquitin, and 100% aa sequence identity shared between human and mouse Ubiquitin (1). In mammals, four Ubiquitin genes encode for two Ubiquitin-ribosomal fusion proteins and two poly-Ubiquitin proteins. Cleavage of the Ubiquitin precursors by deubiquitinating enzymes gives rise to identical Ubiquitin monomers each with a predicted molecular weight of 8.6 kDa. Conjugation of Ubiquitin to target proteins involves the formation of an isopeptide bond between the C-terminal glycine residue of Ubiquitin and a lysine residue in the target protein. This process of conjugation, referred to as ubiquitination or ubiquitylation, is a multi-step process that requires three enzymes: a Ubiquitin-activating (E1) enzyme, a Ubiquitin-conjugating (E2) enzyme, and a Ubiquitin ligase (E3). Ubiquitination is classically recognized as a mechanism to target proteins for degradation and as a result, Ubiquitin was originally named ATP-dependent Proteolysis Factor 1 (APF-1) (2,3). In addition to protein degradation, ubiquitination has been shown to mediate a variety of biological processes such as signal transduction, endocytosis, and post-endocytic sorting (4-7).
This fully functional N-terminal FLAG-tagged Ubiquitin protein allows for the convenient detection or affinity purification of ubiquitinated proteins in vitro. The epitope tag is a hydrophilic octapeptide (DYKDDDDK) derived from the sequence of the bacteriophage 7 gene-10 product. This tag is specifically recognized by Anti-FLAG antibodies M1, M2 or M5.
- Sharp, P.M. & W.-H. Li. (1987) Trends Ecol. Evol. 2:328.
- Ciechanover, A. et al. (1980 ) Proc. Natl. Acad. Sci. USA 77:1365.
- Hershko, A. et al. (1980) Proc. Natl. Acad. Sci. USA 77:1783.
- Greene, W. et al. (2012) PLoS Pathog. 8:e1002703.
- Tong, X. et al. (2012) J. Biol. Chem. 287:25280.
- Wei, W. et al. (2004) Nature 428:194.
- Wertz, I.E. et al. (2004) Nature 430:694.
Citations for Recombinant Human FLAG (DYKDDDDK)-Ubiquitin Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products.The data collected includes not only links to publications in PubMed,but also provides information about sample types, species, and experimental conditions.
13Citations: Showing 1 - 10Filter your results:
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- Ubiquitination of MAP1LC3B by pVHL is associated with autophagy and cell death in renal cell carcinomaAuthors: HM Kang, KH Noh, TK Chang, D Park, HS Cho, JH Lim, CR JungCell Death Dis, 2019;10(4):279.Applications: Bioassay
- Ring finger protein 145 (RNF145) is a ubiquitin ligase for sterol-induced degradation of HMG-CoA reductaseAuthors: LY Jiang, W Jiang, N Tian, YN Xiong, J Liu, J Wei, KY Wu, J Luo, XJ Shi, BL SongJ. Biol. Chem., 2018;0(0):.Applications: Bioassay
- Proteasome-associated HECT-type ubiquitin ligase activity is required for plant immunityAuthors: JJ Furniss, H Grey, Z Wang, M Nomoto, L Jackson, Y Tada, SH SpoelPLoS Pathog., 2018;14(11):e1007447.Species: HumanSample Types: ProteinApplications: Bioassay
- The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor TurnoverAuthors: R Tawo, W Pokrzywa, É Kevei, ME Akyuz, V Balaji, S Adrian, J Höhfeld, T HoppeCell, 2017;169(3):470-482.e13.Applications: Bioassay
- Reciprocal Regulation between 53BP1 and the Anaphase-Promoting Complex/Cyclosome Is Required for Genomic Stability during Mitotic StressAuthors: TJ Kucharski, PE Minshall, M Moustafa-K, AS Turnell, JG TeodoroCell Rep, 2017;18(8):1982-1995.
- Ufd2p synthesizes branched ubiquitin chains to promote the degradation of substrates modified with atypical chainsAuthors: C Liu, W Liu, Y Ye, W LiNat Commun, 2017;8(0):14274.Species: YeastSample Types: Cell LysatesApplications: Bioassay
- COP1 mediates dark-specific degradation of microtubule-associated protein WDL3 in regulating Arabidopsis hypocotyl elongationAuthors: N Lian, X Liu, X Wang, Y Zhou, H Li, J Li, T MaoProc. Natl. Acad. Sci. U.S.A., 2017;114(46):12321-12326.Species: HumanSample Types: Recombinant ProteinApplications: Bioassay
- Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP�NPLOC4�UFD1L is enhanced by a mutation that causes multisystem proteinopathyAuthors: EE Blythe, KC Olson, V Chau, RJ DeshaiesProc. Natl. Acad. Sci. U.S.A., 2017;0(0):.Applications: Bioassay
- Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1Elife, 2016;5(0):.Species: HumanSample Types: ProteinApplications: Ubiquitination
- Dimeric Ube2g2 simultaneously engages donor and acceptor ubiquitins to form Lys48-linked ubiquitin chains.Authors: Liu, Weixiao, Shang, Yonglian, Zeng, Yan, Liu, Chao, Li, Yanchang, Zhai, Linhui, Wang, Pan, Lou, Jizhong, Xu, Ping, Ye, Yihong, Li, WeiEMBO J, 2014;33(1):46-61.Species: N/ASample Types: Recombinant ProteinApplications: Bioassay
- The Rad6/18 ubiquitin complex interacts with the Epstein-Barr virus deubiquitinating enzyme, BPLF1, and contributes to virus infectivity.Authors: Kumar, Ravindra, Whitehurst, Christop, Pagano, Joseph SJ Virol, 2014;88(11):6411-22.Species: N/ASample Types: ProteinApplications: Bioassay
- Cytosolic quality control of mislocalized proteins requires RNF126 recruitment toBag6.Authors: Rodrigo-Brenni M, Gutierrez E, Hegde RMol Cell, 2014;55(2):227-37.Applications: Bioassay
- An unusual two-step control of CPEB destruction by Pin1.Authors: Nechama, Morris, Lin, Chien-Li, Richter, Joel DMol Cell Biol, 2013;33(1):48-58.Species: XenopusSample Types: In VivoApplications: In Vivo
FAQs
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View all Proteins and Enzyme FAQsReconstitution Buffers
Reconstitution Buffer 1 (PBS)
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② 应取pH8.0，这样可使核苷酸带较多负电荷，利于吸附于阴离子交换树脂柱。虽然pH 11.4时核苷酸带有更多的负电荷，但pH过高对分离不利。
③ 当不考虑树脂的非极性吸附时，根据核苷酸负电荷的多少来决定洗脱速度，则洗脱顺序为CMP>AMP> GMP > UMP，但实际上核苷酸和聚苯乙烯阴离子交换树脂之间存在着非极性吸附，嘌呤碱基的非极性吸附是嘧啶碱基的3倍。静电吸附与非极性吸附共同作用的结果使洗脱顺序为：CMP> AMP > UMP >GMP。
1. Buffer中离子浓度过大，甘氨酸或者Tris碱有可能疏忽多加了
2.没有加相应浓度的SDS
3.电泳周围温度高，也是一个原因
我使用的是1%琼脂糖凝胶，电泳缓冲液为0.5×TBE。
请问各位大虾，在不影响实验结果的前提下，这样的含EB的电泳缓冲液可以反复使用多少次？
谢谢^_^
500ml
2。阴极缓冲液 ( 10 × ) ：将 60.55g Tris ,89.58g Tricine 及 5g SDS 溶于400ml 蒸馏水中，加水至终体积为500ml
使用时稀释至1×的缓冲液，电泳槽内槽加入阴极电泳缓冲液，外槽加入阳极电泳缓冲液。形成Trcine-SDS-PAGE电泳系统。
如果你是制胶或者做电泳缓冲液的话，不用灭菌。
Tris-Glycine/SDS;
MOPS/SDS;
Bis-Glycine/SDS等不同的缓冲液、预混液等。
不知道这几种缓冲液有什么异同呢？
比如如果是Invitrogen的预制胶-预混液电泳系统，每种预制胶有适配缓冲液，但是如果用其他的缓冲液似乎也可以得到不错的结果，那么是否说明电泳缓冲液只要满足了缓冲ph范围，其他的不是特别重要呢？
此外，附加提问是，PVDF膜在转膜的时候，转膜缓冲液中不需要加入甲醇，那么是加入甲醇转膜效果好还是不加好呢？
谢谢！
