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.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    使用に関する制限

    法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、 CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、 または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、 いかなる効力も効果も有しません。

    研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。

    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.)