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SUMOylation kit

Market leading kit for generation of SUMOylated proteins in vitro.
BML-UW8955-0001 20 tests 422.00 USD
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  • Fastest assay time on the market, just under 1.5 hours
  • Simple 4 step assay: Mix, Incubate, Quench and Assay
  • High specificity, high throughput capacity
  • 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.
Figure A: Western Blot of SUMOylation Assays for RANGAP1 control target and SP100/p53 target proteins. Assays set-up and run as described in “Assay Protocol”. SUMOylated proteins were detected by Western Blotting on SUMOylation assays containing A: RANGAP1 (Prod. No. BML-KW9755), B: p53 (Prod. No. BML-FW9370) and C: SP100 (Prod. No. BML-UW9825) target proteins with 1: SUMO1 (Prod. No. BML-KW9195), 2: SUMO2 (Prod. No. BML-KW9205) and 3: SUMO3 (Prod. No. BML-KW9215) substrates using the appropriate SUMO antibody (1: Prod. No. BML-PW9460, 2/3:Prod. No. BML- PW9465) as described in “Analysis by Western Blotting”.
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Product Specification

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.
Quantity:Sufficient for 20 assays.
Use/Stability:All kit components should be stored at -80°C to ensure stability and activity.
Handling:Avoid freeze/thaw cycles.
Shipping:Shipped on Dry Ice
Long Term Storage:-80°C
Kit/Set Contains:

20X SUMO Activating Enzyme Solution (SUMO E1) (Prod. No. BML-KW9330)20 µl
20X SUMO Conjugating Enzyme Solution (SUMO E2) (Prod. No. BML-KW9320)20 µl
20X SUMO Enzyme Solutions (SUMO1, SUMO2, SUMO3) (Prod. No. BML-KW9195;  -KW9205; -KW9215)20 µ each
10X SUMOylation Buffer (Prod. No. BML-KW9890)40 µl
20X Control RanGAP1 SUMOylation Target Protein Solution (RG1) (Prod. No. BML-KW9755)20 µl
20X Mg-ATP Solution (Prod. No. BML-KW9805)20 µl
SUMO1 Antibody Solution (Prod. No. BML-PW9460)25 µl; SUMO1 (CT) rabbit polyclonal antibody
SUMO2/3 Antibody Solution (Prod. No. BML-PW9465) 25 µl; SUMO2/3 (NT) rabbit polyclonal antibody


Product Literature References

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;
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.C. 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. Klisczak, et al.; 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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