E2Select Ubiquitin Conjugation Kit Summary
E2Select Ubiquitin Conjugation KitA rapid and economical method to identify E2 enzyme(s) that functionally interact with an E3 ubiquitin ligase in vitro.
Key BenefitsEliminates the need to purchase multiple enzymes individually when attempting to identify which E2 enzyme is involved in a particular ubiquitination reaction.
Kit ContentsEach kit contains reagents sufficient to perform 2 ubiquitination reactions for each E2 enzyme included in the kit.
Principle of the AssayThe kit contains an E2Select plate with a panel of 26 E2 conjugating enzymes arrayed in duplicate on a 96 well plate. This configuration allows an experiment to be performed in duplicate, or the testing of two E3s against a single substrate, or the testing of two substrates with a single E3. Two steps are required to initiate reactions. The first is to assemble all the non-E2 reagents except ATP into a Master Mix and add it to each E2 containing well of the plate (except the positive control wells which already contain these reagents). The second is to add 5 µL ATP to all wells to initiate the ubiquitination reactions. After a 60-minute incubation, the reactions are quenched with 5 µL of 5X Sample Buffer for subsequent analysis by SDS-PAGE/Coomassie or Western blot analysis.
- Add Ubiquitination Master Mix to E2 enzymes arrayed on the plate
- Add ATP
- Incubate at 37˚C for 60 minutes
- Add Sample Buffer
- Analyze by SDS-PAGE and Coomassie or Western blotting
Provided in the kit:
- 96-well plate (contains a panel of 26 E2 conjugating enzymes arrayed in duplicate)
UBE2A UBE2E1 UBE2K UBE2R1 UBE2B UBE2E3 UBE2L3 UBE2R2 UBE2C UBE2DF UBE2N/Uev1a Complex UBE2S UBE2D1 UBE2G1 UBE2N/UBE2V2 Complex UBE2T UBE2D2 UBE2H UBE2Q1-2 UBE2W2 UBE2D3 UBE2J1 UBE2Q2-1 UBE2Z UBE2D4 UBE2J2 - 10X Reaction Buffer
- 10X E1 Enzyme
- 20X Ubiquitin
- 4X Mg2+-ATP
- 5X SDS-PAGE Buffer
- Foil Plate Sealer
Other materials required:
- Sterile Deionized Water (dH2O)
- 1M Dithiothreitol (DTT) – for sample buffer when running reducing SDS-PAGE gels
- SDS-PAGE Gel – 10-20% gradient or others; see product datasheet for guidance
- E3 Ubiquitin Ligase – recombinant or purified from cellular lysates
- Substrate (optional) – not required for analyzing E3 auto-ubiquitination
- Coomassie Stain (optional)
- Western Blot Reagants – transfer buffer, primary and secondary antibodies, nitrocellulose or PVDF membranes, and ECL reagents
- Centrifuge with 96-well plate adapter
- 37° C Incubator
 View Larger Image | Western blot analysis of E2Select plate reactions looking at Ubiquitination of RPN10/S5a mediated by the E3 ligase AIP4/Itch. In vitro reactions using purified E3 Ligase Itch and S5a substrate. Reactions were analyzed by Western Blot Analysis using an anti-S5a primary antibody. N: Control lane with S5a, but no ATP. P: Positive control reaction showing Ubiquitination of S5a. Note the depletion of the unmodified S5a band and the presence of the high molecular weight smear that is Ubiquitinated S5a. Lanes containing a smear or banding pattern above the unmodified S5a band indicate that the E2 is used by Itch in the Ubiquitination process. In this experiment Itch interacted with UBE2D1, -2D2, -2D3, -2D4, UBE2E1, UBE2E3, UBE2L3, and UBE2W2 to promote S5a Ubiquitination to various extents. In some instances cross-reactivity between the antibody and lower molecular species was observed—this can be minimized by using a lower primary antibody concentration. |
Background: Ubiquitin Conjugation KitsE2 conjugating enzymes typically interact with one or two E1 activating enzymes (upstream) and potentially hundreds of E3 ubiquitin ligase enzymes (downstream) to form poly-ubiquitin chains. They connect the activation of ubiquitin to the covalent modification of select lysine residues (ubiquitination) found on target proteins. While E3 ligases play an important role, E2’s are the main determinants in substrate selection and which residues of those substrates become mono- or poly-ubiquitinated. Therefore, they can directly control the cellular fate of the substrate. Mechanistically, E3 ligases bind a "charged" E2-ubiquitin thioester conjugate and catalyze the transfer of ubiquitin from the E2 active site cysteine to a substrate lysine (for RING E3s) or to a catalytic cysteine in the E3 itself (for HECT and RBR E3s).
Due to the complexity of the ubiquitin conjugation system, it can be challenging to determine which E2 conjugating enzyme(s) are utilized by a recently discovered or poorly characterized E3 ligase. The investigator may set up an in vitro ubiquitination reaction using an E1 activating enzyme, Ubiquitin, ATP, and the E3 ligase of interest, but the appropriate E2 for the reaction may not be known. Considering there are approximately 35 active E2 enzymes in humans (15-35 in other eukaryotes), the cost of purchasing each recombinant E2 individually may be cost prohibitive. We offer the E2Select kit as a practical solution to this problem by supplying two reactions worth of a panel of 26 E2 conjugating enzymes contained in a 96 well plate. The kit is designed to help identify which E2 enzymes facilitate in vitro substrate ubiquitination catalyzed by a recombinant E3 ligase and substrate of choice using SDS-PAGE with Coomassie or Western blot analysis.
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. 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). 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.
Specifications
Product Datasheets
Background: Ubiquitin
Reagents Provided
Citations for E2Select Ubiquitin Conjugation Kit
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.
2Citations: Showing 1 - 2Filter your results:
Filter by:
- A tri-ionic anchor mechanism drives Ube2N-specific recruitment and K63-chain ubiquitination in TRIM ligasesAuthors: L Kiss, J Zeng, CF Dickson, DL Mallery, JC Yang, SH McLaughlin, A Boland, D Neuhaus, LC JamesNat Commun, 2019;10(1):4502.2019
- Uncovering and deciphering the pro-invasive role of HACE1 in melanoma cellsAuthors: N El-Hachem, N Habel, T Naiken, H Bzioueche, Y Cheli, GE Beranger, E Jaune, F Rouaud, N Nottet, F Reinier, C Gaudel, P Colosetti, C Bertolotto, R BallottiCell Death Differ., 2018;0(0):.2018
FAQs
No product specific FAQs exist for this product, however you may
View all Activity Assay FAQsReviews for E2Select Ubiquitin Conjugation Kit
There are currently no reviews for this product. Be the first toreview E2Select Ubiquitin Conjugation Kit and earn rewards!
Have you used E2Select Ubiquitin Conjugation Kit?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥1250 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
1、直接法：这是最早的方法。其基本原理是用已知的抗体标记上荧光素后成为特异性荧光抗体，染色时将该抗体直接滴在载玻片上进行孵育，使之直接与载玻片上的抗原结合，在荧光显微镜下直接观察，作出判断。该方法评价：简单易行、特异性高、快速方便，常用于肾活检组织几种免疫球蛋白的检测和病原体的检测。但其不足是只能检测相应的一种物质，敏感性较差，效果有时不理想，目前较少作为更多方面的检测。
2、间接法：该法的基本原理是用特异性的抗体与切片中的抗原结合后，继用间接荧光抗体，与前面的抗原抗体复合物结合，形成抗原抗体荧光复合物。在荧光显微镜下，根据复合物的发光情况来确定所检测的抗原。该方法评价：由于结合在抗原抗体复合物上的荧光素抗体增多，发出的荧光亮度强，因而其敏感性强。目前本法应用较广泛，只需制备一种种属荧光抗体，即可适用于多种第一抗体的标记显示。
3、如果你有针对待查抗原一抗（荧光素标记），而且你的待检抗原表达也比较丰富的话，做直接法也未尝不可。不过如果你不具备上述两个条件的话，还是推荐你做间接法。
4、如果你做的是细胞骨架，那么就是用直接免疫荧光。如果是一般的蛋白表达，这个要看有没有试剂是直接标记的；如果没有，还是只能考虑间接荧光。直接荧光相对间接荧光可以避免非特异姓染色，当然步骤也少了些。我做过细胞骨架的直接染色，效果很好。
这些常用免疫组织化学方法的原理如下：
1. 免疫荧光细胞化学技术
将已知抗体标上荧光素,以此作为探针检查细胞或组织内的相应抗原,在荧光显微镜下观察.当抗原抗体复合物中的荧光素受激发光的照射后会发出一定波长的荧光,从而可以确定组织中的抗原定位或定量.
2. 免疫酶细胞化学技术
是目前免疫组织化学研究中最常用的技术．基本原理是先以酶标记的抗体与组织或细胞作用，然后加入酶的底物，生成有色的不溶性产物或具有一定电子密度的颗粒，通过光镜或电镜，对细胞或组织内的相应抗原进行定位或定性研究．
3. 免疫胶体金技术
就是用胶体金标记一抗，二抗或其他的能特异性的结合免疫球蛋白的分子（如葡萄球菌A蛋白）等作为探针对组织或细胞内的抗原进行定性，定位或定量研究．由于胶体金的电子密度高，多用于免疫电镜的单标记或多标记的定位研究．向左转|向右转
做荧光抗体试验时，定量测定荧光素的含量用到的是哪个波长？
标记免疫技术主要类型：放射免疫技术、酶免疫技术、荧光免疫技术、化学发光免疫技术 基本原理：利用化学或生物发光系统作为抗原抗体反应的指示系统，借以定量检测抗原或抗体的方法，发光物质可直接作为抗原抗体的标记物，也可以游离形式用于催化剂（酶）和辅助剂标记的抗原或抗体的发光反应中。向左转|向右转
免疫组织化学技术按照标记物的种类可分为免疫荧光法、免疫酶法、免疫铁蛋白法、免疫金法及放射免疫自显影法等。免疫荧光细胞化学技术将已知抗体标上荧光素,以此作为探针检查细胞或组织内的相应抗原,在荧光显微镜下观察.当抗原抗体复合物中的荧光素受激发光的照射后会发出一定波长的荧光,从而可以确定组织中的抗原定位或定量.免疫酶细胞化学技术是目前免疫组织化学研究中最常用的技术.基本原理是先以酶标记的抗体与组织或细胞作用，然后加入酶的底物，生成有色的不溶性产物或具有一定电子密度的颗粒，通过光镜或电镜，对细胞或组织内的相应抗原进行定位或定性研究.免疫胶体金技术就是用胶体金标记一抗，二抗或其他的能特异性的结合免疫球蛋白的分子(如葡萄球菌A蛋白)等作为探针对组织或细胞内的抗原进行定性，定位或定量研究.由于胶体金的电子密度高，多用于免疫电镜的单标记或多标记的定位研究。近年来，随着免疫组织化学技术的发展和各种特异性抗体的出现，使许多疑难肿瘤得到了明确诊断。在常规肿瘤病理诊断中，5%-10%的病例单靠H.E.染色难以作出明确的形态学诊断。尤其是免疫组化在肿瘤诊断和鉴别诊断中的实用价值受到了普遍的认可，其在低分化或未分化肿瘤的鉴别诊断时，准确率可达50%-75%。
