Recombinant Human UbcH5a/UBE2D1 Protein, CF Summary
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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.
E2-616
| Formulation | X mg/ml (X μM) in 50 mM HEPES pH 7.5, 200 mM NaCl, 10% (v/v) Glycerol, 1 mM TCEP |
| Shipping | The product is shipped with dry ice or equivalent. 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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Background: UbcH5a/UBE2D1
Ubiquitin-conjugating Enzyme H5a (UbcH5a), also known as Ubiquitin-conjugating Enzyme E2D 1 (UBE2D1), is a ubiquitously expressed protein that is related to Stimulator of Iron Transport (SFT) (1,2). Human UbcH5a/UBE2D1 has a predicted molecular weight of 17 kDa and shares 89% and 88% amino acid (aa) sequence identity with the related family members UbcH5b and UbcH5c, respectively (3). Human UbcH5a/UBE2D1 shares 100% aa sequence identity with the mouse and rat orthologs. UbcH5a/UBE2D1 has a conserved E2 catalytic core domain that contains an active site cysteine residue, and it interacts with a variety of HECT and RING finger Ubiquitin ligases (E3) to mediate the ubiquitination of specific target proteins (4). UbcH5a/UBE2D1 interacts with the E3, E6-AP, to conjugate Ubiquitin to the tumor suppressor, p53 (1). Additional protein targets of UbcH5a/UBE2D1 include c-Fos, RIP1, and HIF-1 (5,6). Pathologically, UbcH5a/UBE2D1 is implicated in protein degradation during cancer and immune responses (7).
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- Gehrke, S. et al. (2003) Blood 101:3288.
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- Lorick, K. et al. (2005) Methods Enzymol. 398:54.
- Stancovski, I. et al. (1995) Mol. Cell Biol. 15:7106.
- Dynek, J. et al. (2010) EMBO J. 29:4128.
- Vanni, E. et al. (2012) J. Virol. 86:6323.
Citations for Recombinant Human UbcH5a/UBE2D1 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.
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- Competitive ubiquitination activates the tumor suppressor p53Authors: X Li, M Guo, L Cai, T Du, Y Liu, HF Ding, H Wang, J Zhang, X Chen, C YanCell Death Differ., 2019;0(0):.Species: HumanSample Types: Recombinant ProteinApplications: Bioassay
- Aryl Sulfonamides Degrade RBM39 and RBM23 by Recruitment to CRL4-DCAF15Authors: TC Ting, M Goralski, K Klein, B Wang, J Kim, Y Xie, D NijhawanCell Rep, 2019;29(6):1499-1510.e6.Species: HumanSample Types: Recombinant ProteinApplications: Bioassay
- Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage responseAuthors: J Sitz, SA Blanchet, SF Gameiro, E Biquand, TM Morgan, M Galloy, J Dessapt, EG Lavoie, A Blondeau, BC Smith, JS Mymryk, CA Moody, A Fradet-TurProc. Natl. Acad. Sci. U.S.A., 2019;116(39):19552-19562.Species: Virus - HPVSample Types: Recombinant ProteinApplications: Bioassay
- Mechanism for recycling tRNAs on stalled ribosomesAuthors: MCJ Yip, AFA Keszei, Q Feng, V Chu, MJ McKenna, S ShaoNat. Struct. Mol. Biol., 2019;0(0):.Species: HumanSample Types: Recombinant ProteinApplications: Ubiquitination
- Suppression of autophagy during mitosis via CUL4-RING ubiquitin ligases-mediated WIPI2 polyubiquitination and proteasomal degradationAuthors: G Lu, J Yi, A Gubas, YT Wang, Y Wu, Y Ren, M Wu, Y Shi, C Ouyang, HWS Tan, T Wang, L Wang, ND Yang, S Deng, D Xia, RH Chen, SA Tooze, HM ShenAutophagy, 2019;0(0):1-18.Applications: Bioassay
- DBC1 Regulates p53 Stability via Inhibition of CBP-Dependent p53 PolyubiquitinationAuthors: OE Akande, PK Damle, M Pop, NE Sherman, BB Szomju, LV Litovchick, SR GrossmanCell Rep, 2019;26(12):3323-3335.e4.Applications: Bioassay
- CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cellsAuthors: S Ito, A Ueno, T Ueda, H Nakagawa, H Taniguchi, N Kayukawa, A Fujihara-I, F Hongo, K Okihara, O UkimuraOncotarget, 2018;9(25):17645-17655.Applications: Bioassay
- E3 ubiquitin ligase RNF123 targets lamin B1 and lamin-binding proteinsAuthors: R Khanna, V Krishnamoo, VK ParnaikFEBS J., 2018;0(0):.Species: HumanSample Types: Recombinant ProteinApplications: Bioassay
- MIB-1 Is Required for Spermatogenesis and Facilitates LIN-12 and GLP-1 Activity inCaenorhabditis elegansAuthors: M Ratliff, KL Hill-Harfe, EJ Gleason, H Ling, TL Kroft, SW L"HernaultGenetics, 2018;0(0):.Species: N/ASample Types: Recombinant ProteinApplications: Bioassay
- Cryo-EM structures and dynamics of substrate-engaged human 26S proteasomeAuthors: Y Dong, S Zhang, Z Wu, X Li, WL Wang, Y Zhu, S Stoilova-M, Y Lu, D Finley, Y MaoNature, 2018;0(0):.Species: HumanSample Types: ProteinApplications: Bioassay
- SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrateAuthors: ME Matyskiela, S Couto, X Zheng, G Lu, J Hui, K Stamp, C Drew, Y Ren, M Wang, A Carpenter, CW Lee, T Clayton, W Fang, CC Lu, M Riley, P Abdubek, K Blease, J Hartke, G Kumar, R Vessey, M Rolfe, LG Hamann, PP ChamberlaiNat. Chem. Biol., 2018;0(0):.Species: HumanSample Types: Recombinant ProteinApplications: Ubiquitination
- Regulation of a distinct activated RIPK1 intermediate bridging complex I and complex II in TNF?-mediated apoptosisAuthors: P Amin, M Florez, A Najafov, H Pan, J Geng, D Ofengeim, SA Dziedzic, H Wang, VJ Barrett, Y Ito, MJ LaVoie, J YuanProc. Natl. Acad. Sci. U.S.A., 2018;115(26):E5944-E5953.Applications: Bioassay
- PI5P4K? functions in DTX1-mediated Notch signalingAuthors: L Zheng, SD ConnerProc. Natl. Acad. Sci. U.S.A., 2018;0(0):.Species: HumanSample Types: Recombinant ProteinApplications: Bioassay
- Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9Authors: CS Yang, K Jividen, A Spencer, N Dworak, L Ni, LT Oostdyk, M Chatterjee, B Ku?mider, B Reon, M Parlak, V Gorbunova, T Abbas, E Jeffery, NE Sherman, BM PaschalMol. Cell, 2017;66(4):503-516.e5.Applications: Bioassay
- DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residuesAuthors: V Glorian, J Allègre, J Berthelet, B Dumetier, PM Boutanquoi, N Droin, C Kayaci, J Cartier, S Gemble, G Marcion, D Gonzalez, R Boidot, C Garrido, O Michaud, E Solary, L DubrezCell Death Dis, 2017;8(5):e2816.Species: Bacteria - E. ColiSample Types: ProteinApplications: Bioassay
- pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4(CRBN) ubiquitin ligaseAuthors: J An, CM Ponthier, R Sack, J Seebacher, MB Stadler, KA Donovan, ES FischerNat Commun, 2017;8(0):15398.Species: N/ASample Types: ProteinApplications: Bioassay
- Thymine DNA glycosylase modulates DNA damage response and gene expression by base excision repair-dependent and independent mechanismsAuthors: T Nakamura, K Murakami, H Tada, Y Uehara, J Nogami, K Maehara, Y Ohkawa, H Saitoh, H Nishitani, T Ono, R Nishi, M Yokoi, W Sakai, K SugasawaGenes Cells, 2017;0(0):.Applications: Bioassay
- Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain UbiquitinationAuthors: Y Lee, TF Chou, SK Pittman, AL Keith, B Razani, CC WeihlCell Rep, 2017;19(1):188-202.Species: MouseSample Types: ProteinApplications: Bioassay
- Ubiquitin Modification by the E3 Ligase/ADP-Ribosyltransferase Dtx3L/Parp9Authors: CS Yang, K Jividen, A Spencer, N Dworak, L Ni, LT Oostdyk, M Chatterjee, B Ku?mider, B Reon, M Parlak, V Gorbunova, T Abbas, E Jeffery, NE Sherman, BM PaschalMol. Cell, 2017;66(4):503-516.e5.Species: HumanSample Types: ProteinApplications: Bioassay
- DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residuesAuthors: V Glorian, J Allègre, J Berthelet, B Dumetier, PM Boutanquoi, N Droin, C Kayaci, J Cartier, S Gemble, G Marcion, D Gonzalez, R Boidot, C Garrido, O Michaud, E Solary, L DubrezCell Death Dis, 2017;8(5):e2816.Species: HumanSample Types: Recombinant ProteinApplications: Bioassay
- Ubiquitin-dependent regulation of Cdc42 by XIAPAuthors: A Murali, J Shin, H Yurugi, A Krishnan, M Akutsu, A Carpy, B Macek, K RajalingamCell Death Dis, 2017;8(6):e2900.Species: HumanSample Types: ProteinApplications: Bioassay
- Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain UbiquitinationAuthors: Y Lee, TF Chou, SK Pittman, AL Keith, B Razani, CC WeihlCell Rep, 2017;19(1):188-202.Species: MouseSample Types: Whole CellsApplications: Bioassay
- Ubiquitin-dependent regulation of Cdc42 by XIAPAuthors: A Murali, J Shin, H Yurugi, A Krishnan, M Akutsu, A Carpy, B Macek, K RajalingamCell Death Dis, 2017;8(6):e2900.Species: N/ASample Types: ProteinApplications: Bioassay
- Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometryAuthors: K Kliza, C Taumer, I Pinzuti, M Franz-Wach, S Kunzelmann, B Stieglitz, B Macek, K HusnjakNat. Methods, 2017;0(0):.Species: N/ASample Types: ProteinApplications: Bioassay
- A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligaseAuthors: Mary E MatyskielaNature, 2016;0(0):.Species: N/ASample Types: ProteinApplications: Bioassay
- Role of SUMO activating enzyme in cancer stem cell maintenance and self-renewalNat Commun, 2016;7(0):12326.Species: N/ASample Types: Recombinant ProteinApplications: Bioassay
- Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activityNucleic Acids Res., 2016;0(0):.Applications: Bioassay
- The adenovirus E4-ORF3 protein functions as a SUMO E3 ligase for TIF-1? sumoylation and poly-SUMO chain elongationProc Natl Acad Sci USA, 2016;113(24):6725-30.Species: HumanSample Types: ProteinApplications: Bioassay
- Pin1 modulates ERalpha levels in breast cancer through inhibition of phosphorylation-dependent ubiquitination and degradation.Authors: Rajbhandari P, Schalper K, Solodin N, Ellison-Zelski S, Ping Lu K, Rimm D, Alarid EOncogene, 2014;33(11):1438-47.Species: N/ASample Types: Recombinant ProteinApplications: Bioassay
- The ubiquitin ligase ASB4 promotes trophoblast differentiation through the degradation of ID2.Authors: Townley-Tilson, W H Davi, Wu, Yaxu, Ferguson, James E, Patterson, CamPLoS ONE, 2014;9(2):e89451.Species: HumanSample Types: Whole CellsApplications: Ubiquitination
- Cellular inhibitor of apoptosis (cIAP)-mediated ubiquitination of phosphofurin acidic cluster sorting protein 2 (PACS-2) negatively regulates tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity.Authors: Guicciardi, Maria Eu, Werneburg, Nathan W, Bronk, Steven F, Franke, Adrian, Yagita, Hideo, Thomas, Gary, Gores, GregoryPLoS ONE, 2014;9(3):e92124.Species: N/ASample Types: Recombinant ProteinApplications: Bioassay
- A HECT ubiquitin-protein ligase as a novel candidate gene for altered quinine andquinidine responses in Plasmodium falciparum.Authors: Sanchez C, Liu C, Mayer S, Nurhasanah A, Cyrklaff M, Mu J, Ferdig M, Stein W, Lanzer MPLoS Genet, 2014;10(5):e1004382.Species: Parasite - Plasmodium falciparumSample Types: ProteinApplications: Bioassay
- C. elegans ring finger protein RNF-113 is involved in interstrand DNA crosslink repair and interacts with a RAD51C homolog.Authors: Lee, Hyojin, Alpi, Arno F, Park, Mi So, Rose, Ann, Koo, Hyeon-SoPLoS ONE, 2013;8(3):e60071.Species: HumanSample Types: Recombinant ProteinApplications: Enzyme Assay
- A novel role for inhibitor of apoptosis (IAP) proteins as regulators ofendothelial barrier function by mediating RhoA activation.Authors: Hornburger M, Mayer B, Leonhardt S, Willer E, Zahler S, Beyerle A, Rajalingam K, Vollmar A, Furst RFASEB J, 2013;28(4):1938-46.Species: HumanSample Types: Recombinant ProteinApplications: Ubiquitination
- Characterization of ML-IAP protein stability and physiological role in vivo.Authors: Varfolomeev, Eugene, Moradi, Elham, Dynek, Jasmin N, Zha, Jiping, Fedorova, Anna V, Deshayes, Kurt, Fairbrother, Wayne J, Newton, Kim, Le Couter, Jennifer, Vucic, DomagojBiochem J, 2012;447(3):427-36.Species: HumanSample Types: ProteinApplications: Bioassay
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因为内参有好几种,分子量差别也比较大,更换分子量与检测蛋白差距更大的内参就可以避免这个问题
另外也可以先用一抗孵育显色和检测,再用Strip缓冲液洗掉膜上的抗体,重新进行内参的抗体孵育显色检测。这样也可以将检测蛋白和内参显色在同一张膜上
【GAPDH】或G3PDH是甘油醛-3-磷酸脱氢酶( glyceraldehyde-3-phosphate dehydrogenase )的英文缩写。GAPDH是参与糖酵解的一种关键酶,由4个30-40kDa的亚基组成,分子量146kDa,检测条带大约在36kDa。GAPDH基因几乎在所有组织中都高水平表达,且GAPDH 作为管家基因在同种细胞或者组织中的蛋白质表达量一般是恒定的。
【Western Blot】蛋白质印迹法(免疫印迹试验)。它是分子生物学、生物化学和免疫遗传学中常用的一种实验方法。其基本原理是通过特异性抗体对凝胶电泳处理过的细胞或生物组织样品进行着色。通过分析着色的位置和着色深度获得特定蛋白质在所分析的细胞或组织中表达情况的信息。
因为这个实验用到了抗体,抗体是特异识别某一种蛋白的,所以只要能检测到信号,说明样品中有该蛋白存在,如果有相应的对照,也能半定量说明该蛋白的量如何。
一般可与大鼠、小鼠、人、兔均有交叉反应,即可作为上述任一来源蛋白的内参。
Actin分子量为42kD,为肌动蛋白
GAPDH分子量为36kD,为磷酸脱氢酶
要检测一个基因的表达产物是否正确,或者比较表达产物量的相对变化,首选方法是Western Blot。因为WesternBlot操作相对简单方便,既可以定性分析表达产物,同时还可以指示目的蛋白量的相对变化。虽然,顺利的时候WesternBlot做起来很简单,可不顺的时候也很令人心烦――做不出结果啦、假阳性啦、结果出现多条带啦、到底是一抗有问题还是二抗有问题啦……毕竟,作为一种有活性的生物大分子,抗体和抗原的反应毕竟不象1+1那么明确,而用这种不确定的试剂来测定同样知之甚少的表达产物,确实是有一定的不确定性的。所以,严谨的WesternBlot实验设计中要求有良好的参照体系,对实验结果分析是非常有用。特别是当实验出现问题时,借助参照体系很容易就可以查出问题所在,而不必抓耳挠腮怨天尤人。良好的参照体系通常包括分子量Marker(用来确定蛋白条带对应的分子量大小),空白载体对照(如果是诱导表达体系还应该有诱导前的对照),已知量标准产物的正对照;另外还有内参。可是由于经费限制或者偷懒的原因,国内的不少人做Western Blot往往省略参照,导致结果出现问题时无法分析结果――即便有结果也可能影响结果的分析。
内参是最容易被忽略的一项。我们知道,要用Western Blot比较不同条件下或者不同组织中,目的蛋白表达量的相对多少,前提条件是等量的细胞上样,才有比较的基础。特别表达量不高时,上样量的差别就很可能影响结果的分析。所以你需要内参。
内参即是内部参照(InternalControl),对于哺乳动物细胞表达来说一般是指由管家基因编码表达的蛋白(HousekeepingProteins),它们在各组织和细胞中的表达相对恒定,在检测蛋白的表达水平变化时常用它来做参照物。在Western Blotting 实验中,除了需要进行蛋白抽提、蛋白定量、等量蛋白上样电泳、转膜、靶蛋白抗体孵育、显色等步骤以外,还需要进行内参的检测,以校正蛋白质定量、上样过程中存在的实验误差,保证实验结果的准确性。
在国外发表的文章中,Western Blotting实验结果须进行内参校正已成为一种惯例。但是,国内仍有不少科研人员在WesternBlotting实验中忽略了内参的使用,将蛋白浓度测定作为规范需相互比较的各种样品间上样量等同的唯一方法。然而各种蛋白质浓度定量方法,都存在局限性,不能完全准确的确定各种样品的准确蛋白浓度。如UV法直接定量,适合测试较纯净、成分相对单一的蛋白质,相对于比色法来说,操作简单,但是容易受到平行物质的干扰,如DNA的干扰;且敏感度低,要求蛋白的浓度较高。比色法测定蛋白浓度一般有BCA,Bradford,Lowry等几种方法。BCA法与Lowry法都容易受到蛋白质之间以及去污剂的干扰。Bradford 法敏感度最高,且与一系列干扰Lowry,BCA反应的还原剂(如DTT,巯基乙醇)相容。但是对于去污剂依然是敏感的,其最主要的缺点是不同的标准品会导致同一样品的结果差异较大,无可比性。另外,蛋白质定量以后进行电泳时需要等量上样,此步骤也存在操作误差。在Western blotting实验时使用内参,即可简便地对定量和上样步骤产生的误差进行校正。
在WesternBlotting中使用内参其实就是在WB过程中的另外用内参对应的抗体检测内参,这样在检测目的产物的同时可以检测内参的表达,由于内参在各组织和细胞中的表达相对恒定,借助检测每个样品内参的量就可以用于校正上样误差,这样半定量的结果才更为可信。此外使用内参可以作为空白对照,检测蛋白转膜情况是否完全、整个Western Blot显色或者发光体系是否正常。
实验结果分析其实很简单:如果样品蛋白量有限,只够进行一次电泳转膜实验时,分别检测样品的内参量和目的蛋白量。将各样品目的蛋白量分别除以其内参含量,得到的数值即为内参校正后的各样品中目的蛋白相对含量,再用此数值进行样品间的比较和分析,得到目的蛋白含量在不同样品间的实际变化结果。如果样品量充分,可以先检测内参,观测样品间内参显色条带是否一致,根据差异大小调整各样品的上样量重新进行Western Blotting实验,至内参量一致为止;若内参一致,即可进行不同样品间目的蛋白表达变化分析。这样虽然麻烦一点,但是可以保证结果更有说服力,更可信。毕竟我们的实验是一种严谨的工作。
附:在Western Blotting实验过程中使用内参的方法有:
一、 超级简便的标记内参使用法:只要在二抗孵育时加入HRP标记内参抗体,按照正常操作即可。
二、普通内参:当目的蛋白的分子量大小与选用的内参蛋白分子量相差不大时,可以先进行目的蛋白的抗体温育显色和检测。然后使用Strip缓冲液洗掉膜上的抗体,重新进行内参蛋白的抗体温育、显色检测。
三、当目的蛋白的分子量大小与选用的内参蛋白分子量大小相差比较明显情况下,可以在转膜后预染,根据蛋白质Marker的大小将膜剪为大分子量和小分子量两部分,使内参蛋白与目的蛋白分开。然后两块膜分别与内参蛋白抗体以及目的蛋白抗体进行温育,二抗温育以及显色。
【摘自网络】展开
β-Actin作为内参是得到了公认的,这是针对大多数组织和细胞来说的,它广泛分布于细胞浆内,表达量非常丰富。Beta-actin由375个氨基酸组成,分子量大小为42-43kDa左右。
β-actin的蛋白水平通常不会发生改变,因此被广泛用于Western时上样量是否一致的参照,也常被用于免疫染色观察细胞的微丝结构。在用作Western的参照时,Actin抗体和Tubulin抗体的主要不同之处在于两者所识别蛋白的分子量不同,这样可以选择合适的参照在同一块胶同一张膜上实现同时检测目标蛋白和参照蛋白。向左转|向右转

