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8692
COX IV (3E11) Rabbit mAb (Alexa Fluor® 594 Conjugate)
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COX IV (3E11) Rabbit mAb (Alexa Fluor® 594 Conjugate) #8692

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Confocal immunofluorescent analysis of HeLa cells using COX IV (3E11) Rabbit mAb (Alexa Fluor® 594 Conjugate) (red) and

β-Actin (13E5) Rabbit mAb (Alexa Fluor® 488 Conjugate) #8844 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Legal Statements:

DRAQ5 is a registered trademark of Biostatus Limited.

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To Purchase # 8692S
Product # Size Price
8692S		 100 µl  (50 tests) $ 305

Custom Formulation

Supporting Data

REACTIVITY H R Mk Z B Pg
SENSITIVITY Endogenous
MW (kDa)
Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 594 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated COX IV (3E11) Rabbit mAb #4850.

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 1:50

Storage:

Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Protocol

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Methanol, 100%
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100.
  4. Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
  5. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.

  1. Aspirate liquid, then cover cells to a depth of 2–3 mm with ice-cold 100% methanol.
  2. Allow cells to fix for 15 minutes at -20°C.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with Immunostaining (Section C).

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  7. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.

posted December 2010

Protocol Id: 221

Specificity / Sensitivity

COX IV (3E11) Rabbit mAb (Alexa Fluor® 594 Conjugate) recognizes endogenous levels of total COX IV protein.

Species Reactivity:

Human, Rat, Monkey, Zebrafish, Bovine, Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys29 of human COX IV protein. Antibodies are purified by protein A and peptide affinity chromatography.

Background

Cytochrome c oxidase (COX) is a hetero-oligomeric enzyme consisting of 13 subunits localized to the inner mitochondrial membrane (1-3). It is the terminal enzyme complex in the respiratory chain, catalyzing the reduction of molecular oxygen to water coupled to the translocation of protons across the mitochondrial inner membrane to drive ATP synthesis. The 3 largest subunits forming the catalytic core are encoded by mitochondrial DNA, while the other smaller subunits, including COX IV, are nuclear-encoded. Research studies have shown that deficiency in COX activity correlates with a number of human diseases (4). The COX IV antibody can be used effectively as a mitochondrial loading control in cell-based research assays.

  1. Ostermeier, C. et al. (1996) Curr. Opin. Struct. Biol. 6, 460-466.
  2. Capaldi, R.A. et al. (1983) Biochim. Biophys. Acta 726, 135-148.
  3. Kadenbach, B. et al. (2000) Free Radic. Biol. Med. 29, 211-221.
  4. Barrientos, A. et al. (2002) Gene 286, 53-63.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc.