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Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 594 Conjugate) #9483
Confocal immunofluorescent analysis of COS-7 cells, transiently transfected with a construct expressing Myc-tagged chorionic somatomammotropin hormone-like 1 (CSL), using Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 594 Conjugate) (red) and β-Catenin (L54E2) Mouse mAb (Alexa Fluor® 488 Conjugate) #2849 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Untransfected cells provide an internal negative control.Learn more about how we get our images
Gallery: Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 594 Conjugate) #9483
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
- 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.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Permeabilization Buffer (1X PBS/0.2% Triton X-100):
To prepare 25 ml, add 2.5 ml 10X PBS, and 22.5 ml dH2O and mix well. While stirring, add 50 µl Triton X-100.
- Image-iT™ FX Signal Enhancer (#11932)
- Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
- 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.
- 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.
- Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
- Allow cells to fix for 15 minutes at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
- Proceed with Immunostaining (Section C).
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.
- Permeabilize the cells in Permeabilization Buffer for 5 minutes at room temperature.
- Rinse three times in 1X PBS for 5 minutes each.
- Apply 3–4 drops of Image-iT™ FX Signal Enhancer (#11932) and incubate for 30 minutes at room temperature.
- Rinse three times with 1X PBS for 5 minutes each.
- Block specimen in Blocking Buffer for 60 minutes.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 minutes each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
- For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.
posted Februay 2011
protocol id: 6
Myc-Tag (9B11) Mouse mAb (Alexa Fluor® 594 Conjugate) detects transfected proteins containing the Myc epitope tag. The antibody recognizes the Myc-tag fused to either the amino or carboxy terminus of targeted proteins in transfected cells.Species Reactivity: All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues 410-419 of human c-Myc (EQKLISEEDL).
Epitope tags are useful for the labeling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Because of their small size, they are unlikely to affect the tagged protein’s biochemical properties.
The Myc epitope tag is widely used to detect expression of recombinant proteins in bacteria, yeast, insect, and mammalian cell systems (1).
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.