Revision 1

#4889Store at -20C

1 Kit

(8 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
Skp1 Antibody 2156 20 µl 19 kDa Rabbit 
Skp2 (D3G5) XP® Rabbit mAb 2652 20 µl 48 kDa Rabbit IgG
ISG15 (22D2) Rabbit mAb 2758 20 µl 15 kDa Rabbit IgG
NEDD8 (19E3) Rabbit mAb 2754 20 µl 9 kDa Rabbit IgG
Ubiquitin (P4D1) Mouse mAb 3936 20 µl Mouse IgG1
UBC3 Antibody 4997 20 µl 32 kDa Rabbit 
SUMO-1 Antibody 4930 20 µl Rabbit 
SUMO-2/3 (18H8) Rabbit mAb 4971 20 µl Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl Horse 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

This sampler kit provides an economical means to investigate protein folding and stability. The kit contains primary and secondary antibodies to perform two Western blots with each antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Background

The small regulatory protein ubiquitin is often covalently linked to many cellular proteins, labeling these targeted proteins for proteasome-mediated degradation. Ubiquitin is first activated by forming a thiolester complex with the E1 activation component. Activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, and then to an E3 ubiquitin ligase for final delivery to the ε-NH2 of the target protein lysine residue (1). The ubiquitin-proteasome pathway has been implicated in a wide range of both normal biological processes and diseases (2,3).
The ubiquitin-like protein family contains three small ubiquitin-related modifier proteins (SUMO-1, -2 and -3), neural precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) and interferon-stimulated 15 kDa protein (ISG15) (4-6). Their covalent attachment to target proteins is a reversible, multi-step process that is analogous to protein ubiquitination. Mature molecules are linked to the activating enzyme E1, conjugated to E2 and ligated to the target proteins by E3 (7-10). Ubiquitin is the predominant regulator for the degradation of a wide range of target proteins (8) while SUMO, NEDD8 and ISG15 modify a limited set of substrates to regulate various other biological processes (4, 11-18).
During ubiquitination, the combinatorial interaction of different E2 and E3 proteins produces variable substrate specificity (4). UBC3 and UBC3B are E2 ubiquitin-carrier proteins (19, 20). The SCF (Skp1/CUL1/F-box) E3 ubiquitin ligase protein complex is composed of three protein components, including the S phase kinase associated protein 1 (Skp1), Cullin homolog 1 (CUL1) and the Skp2 F-box protein (21-23).

  1. Ciechanover, A. (1998) EMBO J. 17, 7151-7160.
  2. Bernardi, R. et al. (2000) Oncogene 19, 2447-2454.
  3. Jesenberger, V. and Jentsch, S. (2002) Nat. Rev. Mol. Cell Biol. 3, 112-121.
  4. Schwartz, D.C. and Hochstrasser, M. (2003) Trends Biochem. Sci. 28, 321-328.
  5. Chiba, T. and Tanaka, K. (2004) Curr. Protein Pept. Sci. 5, 177-184.
  6. Ritchie, K.J. and Zhang, D.E. (2004) Semin. Cell Dev. Biol. 15, 237-246.
  7. Kim, K.I. et al. (2002) J. Cell Physiol. 191, 257-268.
  8. Osaka, F. et al. (1998) Genes Dev. 12, 2263-2268.
  9. Loeb, K.R. and Haas, A.L. (1992) J. Biol. Chem. 267, 7806-7813.
  10. Zhao, C. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 10200-10205.
  11. Matunis, M.J. et al. (1996) J. Cell Biol. 135, 1457-1470.
  12. Duprez, E. et al. (1999) J. Cell Sci. 112 ( Pt 3), 381-393.
  13. Gostissa, M. et al. (1999) EMBO J. 18, 6462-6471.
  14. Rodriguez, M.S. et al. (1999) EMBO J. 18, 6455-6461.
  15. Desterro, J.M. et al. (1998) Mol. Cell 2, 233-239.
  16. Stickle, N.H. et al. (2004) Mol. Cell. Biol. 24, 3251-3261.
  17. Xirodimas, D.P. et al. (2004) Cell 118, 83-97.
  18. Hamerman, J.A. et al. (2002) J. Immunol. 168, 2415-2423.
  19. Semplici, F. et al. (2002) Oncogene 21, 3978-3987.
  20. Pagano, M. et al. (1995) Science 269, 682-685.
  21. Yu, Z.K. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 11324-11329.
  22. Pagano, M. (2004) Mol. Cell 14, 414-416.
  23. Reed, S.I. (2003) Nat. Rev. Mol. Cell Biol. 4, 855-864.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.
    Revision 1
    #4889

    Protein Folding and Stability Antibody Sampler Kit

    Protein Folding and Stability Antibody Sampler Kit: Image 1 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse colon labeled with SUMO-1 Antibody #4930 (left, red). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with β-Catenin (D10A8) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate) #88187 (right, green), Caveolin-1 (D46G3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #31411 (right, gray pseudocolor), and DAPI #4083 (right, blue).
    Protein Folding and Stability Antibody Sampler Kit: Image 2 Expand Image
    Confocal immunofluorescent analysis of fixed frozen mouse CA1 region of hippocampus labeled with SUMO-1 Antibody #4930 (left, red) and co-labeled with Synaptophysin (7H12) Mouse mAb (IF Formulated) #9020 (right, green) and DAPI #4083 (right, blue).
    Protein Folding and Stability Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from PC12 and NIH/3T3 cells, using Skp1 Antibody.
    Protein Folding and Stability Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using Skp2 (D3G5) XP® Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of ubiquitin, NEDD8, ISG15 and SUMO-2/3 recombinant proteins (5 ng each), using NEDD8 (19E3) Rabbit mAb, Ubiquitin (P4D1) Mouse mAb #3936, ISG15 Antibody #2743 and SUMO-2/3 Antibody #4974.
    Protein Folding and Stability Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of lysates from HeLa and A549 cells, untreated (-) or treated with IFN-α (1000 U/mL) (+) for 24 hours, using ISG15 (22D2) Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of 293 and HeLa cells, untreated or treated with the 26S proteasome inhibitor MG132 (50 µM, 90 minutes), using Ubiquitin (P4D1) Mouse mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of recombinant GST-SUMO-1 protein (38 kDa) and extracts from CAD, HeLa and PC-12 cells, using SUMO-1 Antibody.
    Protein Folding and Stability Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of recombinant GST-SUMO-1 protein (38 kDa), recombinant SUMO-2, recombinant SUMO-3, and extracts from C6 and PC12 cells, using SUMO-2/3 (18H8) Rabbit mAb (upper) and SUMO-1 Antibody #4930 (lower).
    Protein Folding and Stability Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from Jurkat, CAD and C6 cells using UBC3 Antibody.
    Protein Folding and Stability Antibody Sampler Kit: Image 11 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    Protein Folding and Stability Antibody Sampler Kit: Image 12 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO* is added and emits light during enzyme catalyzed decomposition.
    Protein Folding and Stability Antibody Sampler Kit: Image 13 Expand Image
    Confocal immunofluorescent images of NIH/3T3 cells labeled with Skp1 Antibody (green, left) compared to an isotype control (right). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Protein Folding and Stability Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a Myc/DDK-tagged cDNA expression construct encoding full-length human Skp2 (hSkp2-Myc/DDK, +), using Skp2 (D3G5) XP® Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of lysates from HeLa, RAW, C6 and COS cells, using NEDD8 (19E3) Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 16 Expand Image
    Immunohistochemical analysis of paraffin-embedded breast carcinoma showing nuclear and cytoplasmic ubiquitin localization, using Ubiquitin (P4D1) Mouse mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using SUMO-1 Antibody.
    Protein Folding and Stability Antibody Sampler Kit: Image 18 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using SUMO-2/3 (18H8) Rabbit mAb (green). Actin filaments were labeled with DyLight® 554 Phalloidin #13054 (red).
    Protein Folding and Stability Antibody Sampler Kit: Image 19 Expand Image
    Flow cytometric analysis of NIH/3T3 cells using Skp1 antibody (blue) compared to a nonspecific negative control antibody (red).
    Protein Folding and Stability Antibody Sampler Kit: Image 20 Expand Image
    Western blot analysis of extracts from HeLa cells expressing either non-targeting shRNA (shNT) or shSkp2, using Skp2 (D3G5) XP® Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    Protein Folding and Stability Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using SUMO-1 Antibody.
    Protein Folding and Stability Antibody Sampler Kit: Image 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Skp2 (D3G5) XP® Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using NEDD8 (19E3) Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma, usinig SUMO-1 Antibody.
    Protein Folding and Stability Antibody Sampler Kit: Image 25 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymphoma using Skp2 (D3G5) XP® Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma, using NEDD8 (19E3) Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 27 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, using SUMO-1 Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 (red).
    Protein Folding and Stability Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Skp2 (D3G5) XP® Rabbit mAb.
    Protein Folding and Stability Antibody Sampler Kit: Image 29 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using NEDD8 (19E3) Rabbit mAb, preincubated with control peptide (left) or Nedd8 Blocking Peptide #1048 (right).
    Protein Folding and Stability Antibody Sampler Kit: Image 30 Expand Image
    Confocal immunofluorescent analyis of HeLa cells expressing either a non-targeting shRNA (shNT; left) or shSkp2 (right) using Skp2 (D3G5) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red) (Cell lines kindly provided by Dr. Wenyi Wei, Harvard Medical School.)
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    Orders: 877-616-CELL (2355)•[email protected]• Support: 877-678-TECH (8324)•[email protected]• Web: cellsignal.com
    For Research Use Only. Not for Use in Diagnostic Procedures.