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4889
Protein Folding and Stability Antibody Sampler Kit

Protein Folding and Stability Antibody Sampler Kit #4889

Western Blotting Image 1

Western blot analysis of extracts from PC12 and NIH/3T3 cells, using Skp1 Antibody.

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Western Blotting Image 2

Western blot analysis of extracts from various cell lines using Skp2 (D3G5) XP® Rabbit mAb.

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Western Blotting Image 3

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.

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Western Blotting Image 4

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.

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Western Blotting Image 5

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.

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Western Blotting Image 6

Western blot analysis of extracts from Jurkat, CAD and C6 cells using UBC3 Antibody.

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Western Blotting Image 7

Western blot analysis of recombinant GST-SUMO-1 protein (38 kDa) and extracts from CAD, HeLa and PC-12 cells, using SUMO-1 Antibody.

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Western Blotting Image 8

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).

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Western Blotting Image 9

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.

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Flow Cytometry Image 10

Flow cytometric analysis of NIH/3T3 cells using Skp1 antibody (blue) compared to a nonspecific negative control antibody (red).

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Western Blotting Image 11

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.

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Western Blotting Image 12

Western blot analysis of lysates from HeLa, RAW, C6 and COS cells, using NEDD8 (19E3) Rabbit mAb.

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IHC-P (paraffin) Image 13

Immunohistochemical analysis of paraffin-embedded breast carcinoma showing nuclear and cytoplasmic ubiquitin localization, using Ubiquitin (P4D1) Mouse mAb.

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Flow Cytometry Image 14

Flow cytometric analysis of untreated Jurkat cells using UBC3 antibody (blue) compared to a nonspecific negative control antibody (red).

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IHC-P (paraffin) Image 15

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using SUMO-1 Antibody.

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IF-IC Image 16

Confocal immunofluorescent analysis of HeLa cells using SUMO-2/3 (18H8) Rabbit mAb (green). Actin filaments were labeled with DyLight® 554 Phalloidin #13054 (red).

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IF-IC Image 17

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).

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Western Blotting Image 18

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).

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IHC-P (paraffin) Image 19

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using NEDD8 (19E3) Rabbit mAb.

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using SUMO-1 Antibody.

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Skp2 (D3G5) XP® Rabbit mAb.

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IHC-P (paraffin) Image 22

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma, using NEDD8 (19E3) Rabbit mAb.

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IHC-P (paraffin) Image 23

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, usinig SUMO-1 Antibody.

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded human lymphoma using Skp2 (D3G5) XP® Rabbit mAb.

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IHC-P (paraffin) Image 25

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using NEDD8 (19E3) Rabbit mAb, preincubated with control peptide (left) or Nedd8 Blocking Peptide #1048 (right).

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IF-IC Image 26

Confocal immunofluorescent analysis of HeLa cells, using SUMO-1 Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 (red).

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IHC-P (paraffin) Image 27

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Skp2 (D3G5) XP® Rabbit mAb.

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Flow Cytometry Image 28

Flow cytometric analysis of untreated HeLa cells using NEDD8 (19E3) Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

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IF-IC Image 29

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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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Skp1 Antibody 2156 20 µl
  • WB
  • IF
  • F
H M R 19 Rabbit 
Skp2 (D3G5) XP® Rabbit mAb 2652 20 µl
  • WB
  • IP
  • IHC
  • IF
H Mk 48 Rabbit IgG
ISG15 (22D2) Rabbit mAb 2758 20 µl
  • WB
  • IP
H 15 Rabbit IgG
NEDD8 (19E3) Rabbit mAb 2754 20 µl
  • WB
  • IP
  • IHC
  • F
H M R Mk 9 Rabbit IgG
Ubiquitin (P4D1) Mouse mAb 3936 20 µl
  • WB
  • IHC
All Mouse IgG1
UBC3 Antibody 4997 20 µl
  • WB
  • F
H M R 32 Rabbit 
SUMO-1 Antibody 4930 20 µl
  • WB
  • IP
  • IHC
  • IF
H M R Mk Rabbit 
SUMO-2/3 (18H8) Rabbit mAb 4971 20 µl
  • WB
  • IF
H M R Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

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.

Skp1 Antibody detects endogenous levels of total Skp1 protein. Skp2 (D3G5) XP® Rabbit mAb recognizes endogenous levels of total Skp2 protein. This antibody is predicted to cross-react with Skp2α and Skp2ß. ISG15 (22D2) Rabbit mAb detects endogenous levels of the uncleaved precursor form of ISG15 protein. This antibody does not recognize the activated (cleaved) or conjugated forms of ISG15. The antibody does not cross-react with other ubiquitin family members, including ubiquitin, SUMO-1, SUMO-2, SUMO-3 and NEDD8. NEDD8 (19E3) Rabbit mAb detects endogenous levels of both free and conjugated NEDD8 protein. The antibody does not cross-react with other ubiquitin family members, including ubiquitin, SUMO-1, SUMO-2, SUMO-3 and ISG15. Ubiquitin (P4D1) Mouse mAb detects ubiquitin, polyubiquitin and ubiquitinated proteins. UBC3 Antibody detects endogenous levels of total UBC3 and UBC3B protein. SUMO-1 Antibody detects recombinant SUMO-1 and endogenous levels of sumoylated proteins (e.g. SUMO-1-RanGAP, 90kD). SUMO-2/3 Antibody detects endogenous levels of SUMO-2/3, but does not cross-react with recombinant SUMO-1.

Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to human SUMO-1 (#4930), UBC3 (#4997), and Skp1 (#2156). Polyclonal antibodies are purified by protein A and peptide affinity chromatography. SUMO-2/3 (18H8) Rabbit mAb (#4971) is produced by immunizing rabbits with a synthetic peptide from the amino terminus of human SUMO-3. Ubiquitin (P4D1) Mouse monoclonal antibody (#3936) is produced by immunizing mice with 1-76 full-length ubiquitin of bovine origin. ISG15 (22D2) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to amino acids at the amino terminus of human ISG15 protein. NEDD8 (19E3) Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to amino acids at the amino terminus of human NEDD8 protein. Skp2 (D3G5) XP® Rabbit mAb is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human Skp2 protein.

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, 7151-60.
  2. Chiba, T. and Tanaka, K. (2004) Curr. Protein Pept. Sci. 5, 177-184.
  3. Ritchie, K.J. and Zhang, D.E. (2004) Semin. Cell Dev. Biol. 15, 237-246.
  4. Pagano, M. (2004) Mol Cell 14, 414-6.
  5. Reed, S.I. (2003) Nat Rev Mol Cell Biol 4, 855-64.
  6. Schwartz, D.C. and Hochstrasser, M. (2003) Trends Biochem Sci 28, 321-8.
  7. Matunis, M.J. et al. (1996) J Cell Biol 135, 1457-70.
  8. Bernardi, R. et al. (2000) Oncogene 19, 2447-54.
  9. Osaka, F. et al. (1998) Genes Dev. 12, 2263-2268.
  10. Loeb, K.R. and Haas, A.L. (1992) J. Biol. Chem. 267, 7806-7813.
  11. Semplici, F. et al. (2002) Oncogene 21, 3978-87.
  12. Kim, K.I. et al. (2002) J. Cell Physiol. 191, 257-68.
  13. Zhao, C. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 10200-10205.
  14. Pagano, M. et al. (1995) Science 269, 682-5.
  15. Jesenberger, V. and Jentsch, S. (2002) Nat Rev Mol Cell Biol 3, 112-21.
  16. Hamerman, J.A. et al. (2002) J. Immunol. 168, 2415-2423.
  17. Stickle, N.H. et al. (2004) Mol. Cell. Biol. 24, 3251-3261.
  18. Duprez, E. et al. (1999) J Cell Sci 112 ( Pt 3), 381-93.
  19. Xirodimas, D.P. et al. (2004) Cell 118, 83-97.
  20. Gostissa, M. et al. (1999) EMBO J 18, 6462-71.
  21. Rodriguez, M.S. et al. (1999) EMBO J 18, 6455-61.
  22. Desterro, J.M. et al. (1998) Mol Cell 2, 233-9.
  23. Yu, Z. K. et al. (1998) Proc Natl Acad Sci. 95, 11324-11329.
Entrez-Gene Id
6233 , 7311 , 7314 , 7316
Swiss-Prot Acc.
P62979 , P62987 , P0CG47 , P0CG48
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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To Purchase # 4889T

Product Number Size Price
4889T 1 Kit (8 x 20 µl) $509.00.0
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