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SUMOylation kit

Market leading kit for generation of SUMOylated proteins in vitro.
 
BML-UW8955-0001 20 tests 390.00 USD
 
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.
bml-uw8955
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: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.
 
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

Small heat shock proteins target mutant CFTR for degradation via a SUMO-dependent pathway: A. Ahner, et al.; Mol. Biol. Cell 2, 74 (2013), 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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