Revision 1

#5142Store at -20C

1 Kit

(5 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
COX IV (3E11) Rabbit mAb 4850 20 µl 17 kDa Rabbit IgG
β-Tubulin (9F3) Rabbit mAb 2128 20 µl 55 kDa Rabbit IgG
Histone H3 (D1H2) XP® Rabbit mAb 4499 20 µl 17 kDa Rabbit IgG
GAPDH (D16H11) XP® Rabbit mAb 5174 20 µl 37 kDa Rabbit IgG
β-Actin (D6A8) Rabbit mAb 8457 20 µl 45 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

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

Description

The Loading Control Antibody Sampler Kit contains antibodies to a variety of housekeeping proteins. The kit contains enough primary and secondary antibodies to perform two western blots per primary 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

Housekeeping proteins perform numerous basic functions within the cell and are constitutively expressed at high levels in a variety of tissues and cell types. Western blot analysis commonly uses housekeeping proteins such as β-actin, COX IV, GAPDH, histone H3 and the α- and β-tubulins as loading controls. Actin is a ubiquitous protein and a major component of the eukaryotic cytoskeleton. Actin exists mainly as the F-actin fibrous polymer (1). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the phosphorylation of glyceraldehyde-3-phosphate during glycolysis. Recent work has demonstrated that GAPDH plays roles in apoptosis (2), gene expression (3), and nuclear transport (4). Globular tubulin subunits made up of α- and β-tubulin heterodimers are the building blocks of microtubules, one of three types of cytosolic fibers that comprise the cytoskeleton (5). Histone proteins, including histone H3, make up the primary building block of chromatin known as nucleosomes. Modulation of the chromatin structure plays an important role in the regulation of transcription in eukaryotes (6). Cytochrome c oxidase (COX) is a hetero-oligomeric enzyme consisting of 13 subunits localized to the inner mitochondrial membrane (7-9). It is the terminal enzyme complex in the respiratory chain, catalyzing the reduction of protons across the mitochondrial inner membrane to drive ATP synthesis (10).

  1. Condeelis, J. (2001) Trends Cell Biol 11, 288-93.
  2. Hara, M.R. and Snyder, S.H. Cell Mol Neurobiol (2006) 26, 527-38.
  3. Zheng, L. et al. (2003) Cell 114, 255-66.
  4. Bae, B.I. et al. (2006) Proc Natl Acad Sci USA 103, 3405-9.
  5. Westermann, S. and Weber, K. (2003) Nat Rev Mol Cell Biol 4, 938-47.
  6. Workman, J.L. and Kingston, R.E. (1998) Annu Rev Biochem 67, 545-79.
  7. Ostermeier, C. et al. (1996) Curr Opin Struct Biol 6, 460-6.
  8. Capaldi, R.A. et al. (1983) Biochim Biophys Acta 726, 135-48.
  9. Kadenbach, B. et al. (2000) Free Radic Biol Med 29, 211-21.
  10. Barrientos, A. et al. (2002) Gene 286, 53-63.

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.

    Limited Uses

    Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

    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.

    Revision 1
    #5142

    Loading Control Antibody Sampler Kit

    Loading Control Antibody Sampler Kit: Image 1 Expand Image
    Simple Western analysis of lysates (1.0 mg/mL) from COS-7 cells using COX IV (3E11) Rabbit mAb #4850. The virtual lane view (left) shows the target bands (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.
    Loading Control Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from HeLa cells using GAPDH (D16H11) XP ® Rabbit mAb #5174. The virtual lane view (left) shows a single 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.
    Loading Control Antibody Sampler Kit: Image 3 Expand Image
    Western blot analysis of extracts from COS-7, NIH/3T3 and PC12 cells, using β-Tubulin (9F3) Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using Histone H3 (D1H2) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 5 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Histone H3 (D1H2) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from HeLa, Jurkat and COS cell lines, using COX IV (3E11) Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from various cell lines using GAPDH (D16H11) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 8 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.
    Loading Control Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from various cell lines using β-Actin (D6A8) Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 10 Expand Image
    Immunohistochemical analysis of paraffin-embedded human glioblastoma using β-Tubulin (9F3) Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 11 Expand Image
    Immunohistochemical analysis of paraffin-embedded 4T1 syngeneic mammary tumor using Histone H3 (D1H2) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 12 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing staining of the mitochondria, using COX IV (3E11) Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 13 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using GAPDH (D16H11) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 14 Expand Image
    Western blot analysis of recombinant Actin isoforms using β-Actin (D6A8) Rabbit mAb (upper) and Pan-Actin Antibody #4968 (lower).
    Loading Control Antibody Sampler Kit: Image 15 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using β-Tubulin (9F3) Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 16 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using Histone H3 (D1H2) XP® Rabbit mAb (green) and β-Tubulin (9F3) Rabbit mAb (Alexa Fluor® 555 Conjugate) #2116 (red).
    Loading Control Antibody Sampler Kit: Image 17 Expand Image
    Immunohistochemical analysis of paraffin-embedded LL/2 syngeneic tumor using Histone H3 (D1H2) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using COX IV (3E11) Rabbit mAb in the presence of control peptide (left) or Cox IV Blocking Peptide #1034 (right).
    Loading Control Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse colon using GAPDH (D16H11) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using β-Actin (D6A8) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Loading Control Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human melanoma using β-Tubulin (9F3) Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 22 Expand Image
    Flow cytometric analysis of HeLa cells using Histone H3 (D1H2) XP® Rabbit mAb (solid line) or a 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.
    Loading Control Antibody Sampler Kit: Image 23 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse brain using Histone H3 (D1H2) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded H1650 xenograft, using COX IV Rabbit mAb. Note specific staining of human cancer cells.
    Loading Control Antibody Sampler Kit: Image 25 Expand Image
    Confocal immunofluorescent analysis of C2C12 cells using GAPDH (D16H11) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Loading Control Antibody Sampler Kit: Image 26 Expand Image
    Flow cytometric analysis of HeLa cells using β-Actin (D6A8) 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.
    Loading Control Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using β-Tubulin (9F3) Rabbit mAb preincubated with control peptide (left) or β-Tubulin Blocking Peptide #1032 (right).
    Loading Control Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded rhesus monkey liver using Histone H3 (D1H2) XP® Rabbit mAb.
    Loading Control Antibody Sampler Kit: Image 29 Expand Image
    Confocal immunofluorescent analysis of HeLa cells using β-Tubulin (9F3) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    Loading Control Antibody Sampler Kit: Image 30 Expand Image
    Confocal immunofluorescent analysis of HeLa cells labeled with COX IV (3E11) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    Loading Control Antibody Sampler Kit: Image 31 Expand Image

    Flow cytometric analysis of K-562 cells using β-Tubulin (9F3) 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.

    Loading Control Antibody Sampler Kit: Image 32 Expand Image
    Flow cytometric analysis of HeLa cells using COX IV (3E11) 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.