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Fastest assay time on the market, just under 1.5 hours
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Simple 4 step assay: Mix, Incubate, Quench and Assay
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High specificity, high throughput capacity
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Amenable to analysis via Western blotting or proteomic methods
This kit provides a means of generating SUMOylated proteins in vitro, by covalent linkage of the carboxy-terminal of SUMO-1, -2 or -3 to specific lysine residues on the target protein via isopeptide bonds, using the SUMOylation enzyme cascade. A short sequence containing the consensus ?-K-X-D/E (where lysine is the amino acid modified, ? is a large hydrophobic residue and X is any amino acid residue) is thought to be necessary for this in vitro protein SUMOylation to occur, however SUMOylation has also been observed in cases where the consensus site is not conserved. A control target protein is provided together with all other necessary components. SUMO specific antibodies are provided for detection of SUMOylated proteins via SDS-PAGE and Western blotting. Provides sufficient material for 20 x 20µL reactions. Suggested uses: For SUMO-modification of specific proteins in vitro, To demonstrate that novel proteins are potential targets for SUMOylation under in vitro conditions, To generate substrates for deSUMOylating enzymes, such as SENP1 and SENP2, To test proteins for SUMO E3 ligase activity.
Western Blot of SUMOylation Assays for control RanGAP1 target protein. Assays set-up and run as described in “Assay Protocol”. SUMOylated proteins were detected by Western Blotting as described in “Analysis by Western Blotting”. B: detected by SUMO-2/3, pAb (BML-PW9465).
Western Blot of SUMOylation Assays for control RanGAP1 target protein. Assays set-up and run as described in “Assay Protocol”. SUMOylated proteins were detected by Western Blotting as described in “Analysis by Western Blotting”. A: detected by SUMO-1, pAb (BML-PW8330)
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Product Details
| Application Notes: | Uses:
1. SUMO-modification of specific proteins in vitro. Allow investigation of the effect SUMOylation has on enzyme function, stabilisation, protein:protein interactions and, hence, it’s role in regulation of cellular processes, such as the p53 tumour repressor and NF-κB pathways.
2. Demonstrate novel proteins are potential targets for SUMOylation under in vitro conditions. Starting point for examining the role SUMOylation of a protein might play in vivo.
3. Generate substrates for deSUMOylating enzymes, such as SENP1 (Prod. No. BML-UW9760) and SENP2 (Prod. No. BML-UW9765).
4. Test proteins for SUMO E3 ligase activity: does it facilitate or enhance SUMOylation of specific target proteins, particularly under conditions/enzyme concentrations that more closely represent those in vivo.
5. Addition of known SUMO E3 ligase to facilitate/enhance target protein SUMOylation, particularly under conditions/enzyme concentrations that more closely represent those in vivo (e.g. RANBP2 [Prod. No. BML-UW9455], shown to be a ligase for SP100 SUMOylation).
6. SUMOylation of proteins in cell lysates or crude fractions/preparations to facilitate investigation of their role/function in complex solutions.
7. Demonstrate SUMOylation of known proteins in specific lysates (confirm with target protein specific antibodies).
8. Use of cell lysate or crude fractions/preparations as source of SUMO E3 ligases to facilitate SUMOylation of purified target proteins in the presence of SUMOylation kit components.
Note: Protocol provided for application 1. Assay set-up can be readily modified for alternative applications by inclusion, omission or substitution of specific components. |
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| Quantity: | Sufficient for 20 assays. |
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| Use/Stability: | Unopened kit should be stored at -80°C to ensure stability and activity. After opening, The SUMO antibody solutions (BML-PW8330 and BML-PW9460) can be stored at -20°C or -80°C. Other components should be stored at -80°C. |
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| Handling: | Avoid freeze/thaw cycles. |
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| Shipping: | Dry Ice |
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| Long Term Storage: | -80°C |
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| Contents: | 20x SUMO Activating Enzyme Solution (SUMO E1): SUMO activating enzyme E1 (human), (recombinant), (Prod. No. BML-UW9330), 20 µl.
20x SUMO Conjugating Enzyme Solution (SUMO E2): Ubc9 (human), (recombinant) (untagged), (Prod. No. BML-UW9320), 20 µl.
20x SUMO Enzyme Solutions (SUMO-1, SUMO-2, SUMO-3): SUMO-1 (human), (reccombinant) (His-tag), (Prod. No. BML-UW9195), 20 µl;
SUMO-2 (human), (reccombinant) (His-tag), (Prod. No. BML-UW9205), 20 µl;
SUMO-3 (human), (reccombinant) (His-tag), (Prod. No. BML-UW9215), 20 µl.
10x SUMOylation Buffer (Prod. No. BML-KW9890), 40 µl
20x Control RanGAP1 SUMOylation Target Protein Solution: RanGAP1 fragment (human), (recombinant) (GST-tag), (Prod. No. BML-UW9755), 20 µl.
20x Mg-ATP Solution (Prod. No. BML-EW9805), 25 µl
SUMO Antibody Solutions:
SUMO-1 (human) (NT), polyclonal antibody, (Prod. No. BML-PW8330), 25 µl;
SUMO-2/3 (human) (NT), polyclonal antibody, (Prod. No. BML-PW9465), 25 µl. |
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| Regulatory Status: | RUO - Research Use Only |
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Product Literature References
FOXK2 affects cancer cell response to chemotherapy by promoting nucleotide de novo synthesis: Y. Li, et al.; Drug Resist. Updat.
67, 100926 (2023),
Abstract;
SUMOylation-mediated PSME3-20S proteasomal degradation of transcription factor CP2c is crucial for cell cycle progression: S.H. Son, et al.; Sci. Adv.
9, eadd4969 (2023),
Abstract;
FGFR1 SUMOylation coordinates endothelial angiogenic signaling in angiogenesis: X. Zhou, et al.; PNAS
119, e2202631119 (2022),
Abstract;
TRPV1 SUMOylation suppresses itch by inhibiting TRPV1 interaction with H1 receptors: Y. Gao, et al.; Cell Rep.
39, 110972 (2022),
Abstract;
SENP1-mediated deSUMOylation of JAK2 regulates its kinase activity and platinum drug resistance: J. Li, et al.; Cell Death Dis.
12, 41419 (2021),
Abstract;
DUSP6 SUMOylation protects cells from oxidative damage via direct regulation of Drp1 dephosphorylation: R. Ma, et al.; Sci. Adv.
6, eaaz0361 (2020),
Abstract;
Full Text
Mitochondrial MUL1 E3 ubiquitin ligase regulates Hypoxia Inducible Factor (HIF-1α) and metabolic reprogramming by modulating the UBXN7 cofactor protein: L. Cilenti, et al.; Sci. Rep.
10, 1609 (2020),
Abstract;
Full Text
Stability of Begomoviral pathogenicity determinant βC1 is modulated by mutually antagonistic SUMOylation and SIM interactions: A. Nair, et al.; BMC Biol.
18, 110 (2020),
Abstract;
Full Text
TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks: M. Li, et al.; PNAS
117, 23588 (2020),
Abstract;
Full Text
ZZW-115–dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents: W. Lan, et al.; JCI Insight
5, e138117 (2020),
Abstract;
Full Text
A SUMOylation-dependent switch of RAB7 governs intracellular life and pathogenesis of Salmonella Typhimurium: G. Mohapatra, et al.; J. Cell Sci.
132, jcs222612 (2019),
Abstract;
The axonal motor neuropathy-related HINT1 protein is a zinc- and calmodulin-regulated cysteine SUMO protease: E. Cortes-Montero, et al.; Antioxid. Redox Signal.
31, 503 (2019),
Abstract;
Heterochromatin protects retinal pigment epithelium cells from oxidative damage by silencing p53 target genes: L. Gong, et al.; PNAS
115, e3987 (2018),
Abstract;
Full Text
RanBP2 regulates the anti-retroviral activity of TRIM5α by SUMOylation at a predicted phosphorylated SUMOylation motif: G. Maarifi, et al.; Commun. Biol.
1, 193 (2018),
Abstract;
Full Text
RAS GTPases are modified by SUMOylation: B.H. Choi, et al.; Oncotarget
9, 4440 (2018),
Abstract;
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SUMO targeting of a stress-tolerant Ulp1 SUMO protease: J. Peek, et al.; PLoS One
13, e0191391 (2018),
Abstract;
Full Text
SUMOylation regulates cytochrome P450 2E1 expression and activity in alcoholic liver disease: M.L. Tomasi, et al.; FASEB J.
32, 3278 (2018),
Abstract;
Full Text
Temporal and SUMO-specific SUMOylation contribute to the dynamics of Polo-like kinase 1 (PLK1) and spindle integrity during mouse oocyte meiosis: W.B. Feitosa, et al.; Dev. Biol.
434, 278 (2018),
Abstract;
Sam68 Allows Selective Targeting of Human Cancer Stem Cells: Y.D. Benoit, et al.; Cell Chem. Biol.
24, 833 (2017),
Abstract;
TRIB3 Promotes APL Progression through Stabilization of the Oncoprotein PML-RARα and Inhibition of p53-Mediated Senescence: K. Ki, et al.; Cancer Cell
31, 697 (2017),
Abstract;
FSCB phosphorylation regulates mouse spermatozoa capacitation through suppressing SUMOylation of ROPN1/ROPN1L: X. Zhang, et al.; Am. J. Transl. Res.
8, 2776 (2016),
Application(s): In vitro SUMOylation assay,
Abstract;
Full Text
SUMO-modification of the La protein facilitates binding to mRNA in vitro and in cells: V. Kota, et al.; PLoS One.
11, e0156365 (2016),
Abstract;
Full Text
SUMOylation of large tumor suppressor 1 at Lys751 attenuates its kinase activity and tumor-suppressor functions: L. Mei, et al.; Cancer Lett.
386, 1 (2016),
Abstract;
ATPase-Dependent Control of the Mms21 SUMO Ligase during DNA Repair: M. Bermúdez-López, et al.; PLoS Biol
13, e1002089 (2015),
Application(s): Assay,
Abstract;
Full Text
Deubiquitinating activity of CYLD is impaired by SUMOylation in neuroblastoma cells: T. Kobayashi, et al.; Oncogene
34, 2251 (2015),
Application(s): In vitro SUMOylation of 6His-tagged CYLD ,
Abstract;
Methionine adenosyltransferase α2 sumoylation positively regulate Bcl-2 expression in human colon and liver cancer cells: M.L. Tomasi, et al.; Oncotarget
6, 37706 (2015),
Abstract;
Full Text
MYCBP2 Is a guanosine exchange factor for Ran protein and determines its localization in neurons of dorsal root ganglia: A. Dörr, et al.; J. Biol. Chem.
290, 25620 (2015),
Abstract;
SENP2 regulates MMP13 expression in a bladder cancer cell line through SUMOylation of TBL1/TBLR1: M. Tan, et al.; Sci. Rep.
5, 13996 (2015),
Abstract;
Full Text
SUMOylation Blocks the Ubiquitin-Mediated Degradation of the Nephronophthisis Gene Product Glis2/NPHP7: H. Ramachandran, et al.; PLoS One
10, e0130275 (2015),
Application(s): Assay using HEK 293T cells,
Abstract;
Full Text
UBC9-dependent association between calnexin and protein tyrosine phosphatase 1B (PTP1B) at the endoplasmic reticulum: D. Lee, et al.; J. Biol. Chem.
290, 5725 (2015),
Abstract;
Full Text
Ehrlichia chaffeensis exploits host SUMOylation pathways to mediate effector-host interactions and promote intracellular survival: P.S. Dunphy, et al.; Infect Immun.
82, 4154 (2014),
Abstract;
FOXL2 posttranslational modifications mediated by GSK3β determine the growth of granulosa cell tumours: J.H. Fox, et al.; Nat. Commun.
5, 2936 (2014),
Abstract;
PIASy-mediated sumoylation of SREBP1c regulates hepatic lipid metabolism upon fasting signaling: G.Y. Lee, et al.; Mol. Cell. Biol.
34, 926 (2014),
Application(s): In vitro SUMOylation of SREBP1c,
Abstract;
Full Text
SUMOylation determines turnover and localization of nephrin at the plasma membrane: I. Tossidou, et al.; Kidney Int.
86, 1161 (2014),
Abstract;
SUMOylation inhibits FOXM1 activity and delays mitotic transition: S.S. Myatt, et al.; Oncogene
33, 4316 (2014),
Application(s): In vitro sumoylation of recombinant FOXM1,
Abstract;
Full Text
Visualizing and quantifying protein polySUMOylation at the single-molecule level: Y. Yang, et al.; Anal. Chem.
86, 967 (2014),
Abstract;
Regulation of stress-inducible phosphoprotein 1 nuclear retention by protein inhibitor of activated STAT PIAS1: I.N. Soares, et al.; Mol. Cell. Proteomics
12, 3253 (2013),
Application(s): In vitro sumoylation of mouse STI1 and human RanGAP1 ,
Abstract;
Full Text
Small heat shock proteins target mutant CFTR for degradation via a SUMO-dependent pathway: A. Ahner, et al.; Mol. Biol. Cell
2, 74 (2013),
Abstract;
Full Text
SUMO modification of menin: Z.J. Feng, et al.; Am. J. Cancer Res.
3, 96 (2013),
Application(s): In vitro SUMOylation of menin,
Abstract;
Full Text
Chemotherapeutic sensitivity of testicular germ cell tumors under hypoxic conditions is negatively regulated by SENP1-controlled sumoylation of OCT4: Y. Wu, et al.; Cancer Res.
72, 4963 (2012),
Abstract;
Forkhead box protein A2 (FOXA2) protein stability and activity are regulated by sumoylation: N.S. Belaguli, et al.; PLoS One
7, e48019 (2012),
Application(s): In vitro sumoylation of FOXA2 or FOXA2K6R mutant proteins ,
Abstract;
Full Text
Identification of sumoylation sites in CCDC6, the first identified RET partner gene in papillary thyroid carcinoma, uncovers a mode of regulating CCDC6 function on CREB1 transcriptional activity: C. Luise, et al.; PLoS One
7, e49298 (2012),
Application(s): In vitro sumoylation of whole cell lysates and recombinant CCDC6 protein,
Abstract;
Full Text
Post-translational modification of the RhoGTPase activating protein 21, ARHGAP21, by SUMO2/3: C.L. Bigarella, et al.; FEBS Lett.
586, 3522 (2012),
Application(s): In vitro sumoylation of ARHGAP21 protein,
Abstract;
SUMO ligase activity of vertebrate Mms21/Nse2 is required for efficient DNA repair but not for Smc5/6 complex stability: M. Kliszczak; DNA Repair (Amst.)
11, 799 (2012),
Application(s): In vitro sumoylation of Nse2 protein,
Abstract;
SUMOylation of the small GTPase ARL-13 promotes ciliary targeting of sensory receptors: Y. Li, et al.; J. Cell Biol.
199, 589 (2012),
Abstract;
The human cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNA-dependent manner: E. Sinigalia, et al.; PLoS One
7, e49630 (2012),
Application(s): In vitro sumoylation of UL44 protein,
Abstract;
Full Text
Sumoylation and nuclear translocation of S100A4 regulate IL-1beta-mediated production of matrix metalloproteinase-13: K.J. Miranda, et al.; J. Biol. Chem.
285, 31517 (2010),
Application(s): In vitro sumoylation of purified recombinant human S100A4 protein ,
Abstract;
Full Text
Sumoylation of forkhead L2 by Ubc9 is required for its activity as a transcriptional repressor of the Steroidogenic Acute Regulatory gene: F.T. Kuo, et al.; Cell. Signal.
21, 1935 (2009),
Abstract;
Full Text
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