Revision 1

#9328Store at -20C

1 Kit

(9 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
c-Fos Antibody 4384 20 µl 62 kDa Rabbit 
c-Abl Antibody 2862 20 µl 135 (c-Abl); 210 (Bcr-Abl) kDa Rabbit 
c-Jun (60A8) Rabbit mAb 9165 20 µl 43, 48 kDa Rabbit IgG
c-Kit (D13A2) XP® Rabbit mAb 3074 20 µl 120 and 145 kDa Rabbit 
c-Myc (D84C12) Rabbit mAb 5605 20 µl 57-65 kDa Rabbit IgG
c-Raf Antibody 9422 20 µl 65 to 75 kDa Rabbit 
Ras (27H5) Rabbit mAb 3339 20 µl 21 kDa Rabbit IgG
Src (32G6) Rabbit mAb 2123 20 µl 60 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 
c-Rel (D4Y6M) Rabbit mAb 12707 20 µl 68-78 kDa Rabbit IgG

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

Description

The c-Oncogene Antibody Sampler Kit provides an economical means of evaluating total levels of various oncogenic proteins. The kit contains enough primary and secondary antibodies to perform two Western blot experiments.

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 regulation of cell growth, differentiation and programmed death is coordinated by several sets of proteins that comprise essential signal transduction pathways. Many of these key regulatory proteins are encoded by proto-oncogenes, which can be activated (altered) to change the typical cell program to one of abnormal cell growth and unregulated development. Proteins encoded by proto-oncogenes include growth factors and other ligands, receptor proteins, tyrosine kinases, various regulatory proteins (i.e. GTPases) and transcription factors. Together these proteins comprise the basic elements of cell signaling pathways; altered expression or mutation of one or more of these components can lead to oncogenic growth (reviewed in 1).
Non-receptor (i.e. cytoplasmic, nuclear) tyrosine kinases such as c-Abl and Src play key roles in the regulation of cell proliferation, differentiation, apoptosis, cell adhesion and stress responses (2,3). Alteration of the corresponding c-Abl and Src proto-oncogenes is associated with oncogenesis; Abl1-BCR gene translocations result in chronic myelogenous leukemia (CML) while constitutively active Src is seen in some patients with colon cancer and altered Src expression is seen in a wide array of cancers (2,4). Regulation of Raf tyrosine kinase by Ras GTPase controls downstream kinases in the MEK/MAPK signaling pathway (5). Activation of the Ras and Raf proto-oncogenes are common in human cancers and both proteins are seen as potential therapeutic targets (6). The receptor tyrosine kinase c-Kit plays a critical role in activation and growth of hematopoietic stem cells (7); mutations that inhibit c-Kit kinase activity are associated with a variety of developmental disorders while mutations producing constitutively active c-Kit can result in mastocytosis and gastrointestinal stromal tumors (8). The alteration of key transcription factors such as c-Fos, c-Jun, c-Myc and c-Rel that are normally responsible for regulating cell and tissue growth, differentiation and the inflammation/immune response, can also result in unregulated, oncogenic cell growth (9-12).

  1. Croce, C.M. (2008) N Engl J Med 358, 502-11.
  2. Wang, J.Y. (2000) Oncogene 19, 5643-50.
  3. Thomas, S.M. and Brugge, J.S. (1997) Annu Rev Cell Dev Biol 13, 513-609.
  4. Dehm, S.M. and Bonham, K. (2004) Biochem Cell Biol 82, 263-74.
  5. Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
  6. Stites, E.C. et al. (2007) Science 318, 463-7.
  7. Gommerman, J.L. et al. (1997) J Biol Chem 272, 30519-25.
  8. Nocka, K. et al. (1990) EMBO J 9, 1805-13.
  9. Milde-Langosch, K. (2005) Eur J Cancer 41, 2449-61.
  10. Shaulian, E. and Karin, M. (2002) Nat Cell Biol 4, E131-6.
  11. Yokota, J. et al. (1986) Science 231, 261-5.
  12. Rayet, B. and Gélinas, C. (1999) Oncogene 18, 6938-47.

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.

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    Revision 1
    #9328

    c-Oncogene Antibody Sampler Kit

    c-Oncogene Antibody Sampler Kit: Image 1 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from K-562 cells using c-Abl Antibody #2862. The virtual lane view (left) shows the target bands (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66 - 440 kDa separation module.
    c-Oncogene Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (1 mg/mL) from Raji cells using c-Myc (D84C12) Rabbit mAb #5605. The virtual lane view (left) shows a single target band (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    c-Oncogene Antibody Sampler Kit: Image 3 Expand Image

    Simple Western™ analysis of lysates (0.1 mg/mL) from HeLa cells treated with UV (100 mJ/cm2; 2H recovery) using c-Jun (60A8) Rabbit mAb #9165. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.

    c-Oncogene Antibody Sampler Kit: Image 4 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from HeLa cells serum starved for 24hr and treated with TPA (400nM, 4hr) using c-Raf Antibody #9422. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    c-Oncogene Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell lines and tissues using c-Rel (D4Y6M) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of cell extracts from various cell lines, using Src (32G6) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines, using c-Abl Antibody.
    c-Oncogene Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from NCI-H526 cells using c-Kit (D13A2) XP® Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from 293T, C2C12 and C6 cells using Ras (27H5) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from HeLa, RAW, and H-4-IIE cells serum-starved overnight and TPA-stimulated for 4 hours, using c-Fos Antibody.
    c-Oncogene Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from control HEK293 cells (lane 1) or c-Myc knockout HEK293 cells (lane 2) using c-Myc (D84C12) Rabbit mAb Antibody, #5605 (upper) or β-actin (13E5) Rabbit mAb, #4970 (lower). The absence of signal in the c-Myc knockout HEK293 cells confirms specificity of the antibody for c-Myc.
    c-Oncogene 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.
    c-Oncogene Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from control HeLa cells (lane 1) or c-Jun knockout HeLa cells (lane 2) using c-Jun (60A8) Rabbit mAb #9165. The absence of signal in the c-Jun knockout HeLa cells confirms specificity of the antibody for c-Jun.
    c-Oncogene Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of extracts from HeLa, C2C12 or PC12 cells, untreated or TPA-treated (200 nM for 30 minutes), using c-Raf Antibody #9422.
    c-Oncogene Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from Neuro-2a cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® c-Rel siRNA I (Mouse Specific) #13058 (+) or SignalSilence® c-Rel siRNA II (Mouse Specific) #13170 (+), using c-Rel (D4Y6M) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The c-Rel (D4Y6M) Rabbit mAb confirms silencing of c-Rel expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.
    c-Oncogene Antibody Sampler Kit: Image 16 Expand Image
    Confocal immunofluorescent analysis of NCI-H526 (left) and Jurkat (right) cells using c-Kit (D13A2) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    c-Oncogene Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of extracts from HeLa cells, mock transfected or transfected with SignalSilence® c-Myc siRNA I #6341, using c-Myc (D84C12) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 18 Expand Image
    Western blot analysis of extracts from NIH/3T3 and SK-N-MC cells, untreated or UV-treated, using c-Jun (60A8) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 19 Expand Image
    Western blot analysis of extracts from various cell lines using c-Myc (D84C12) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded human astrocytoma, using c-Jun (60A8) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 21 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, mock-transfected (left) or transfected with SignalSilence® c-Myc siRNA I #6341 (right), using c-Myc (D84C12) Rabbit mAb (green). Actin filaments have been labeled wth DY-554 phalloidin (red).
    c-Oncogene Antibody Sampler Kit: Image 22 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, using c-Jun (60A8) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma, using c-Jun (60A8) Rabbit mAb.
    c-Oncogene Antibody Sampler Kit: Image 24 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, using c-Jun (60A8) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).
    c-Oncogene Antibody Sampler Kit: Image 25 Expand Image
    Flow cytometric analysis of Jurkat cells using c-Jun (60A8) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    c-Oncogene Antibody Sampler Kit: Image 26 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from PC-12 cells starved overnight and treated with β-NGF #5221 (50ng/ml) for 2h and c-Jun (60A8) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across Dclk1, a known target gene of c-Jun (see additional figure containing ChIP-qPCR data).
    c-Oncogene Antibody Sampler Kit: Image 27 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from PC-12 cells starved overnight and treated with β-NGF #5221 (50ng/ml) for 2h and c-Jun (60A8) Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 2 (upper), including Dclk1 (lower), a known target gene of c-Jun (see additional figure containing ChIP-qPCR data).
    c-Oncogene Antibody Sampler Kit: Image 28 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from PC-12 cells starved overnight and treated with β-NGF #5221 (50ng/ml) for 2h and either of c-Jun (60A8) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Rat CCRN4L Promoter Primers #7983, rat DCLK1 promoter primers, and SimpleChIP® Rat GAPDH Promoter Primers #7964. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.