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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Endogenous Rabbit IgG
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Flow Cytometry

Flow cytometric analysis of Jurkat cells using c-Myc (D84C12) Rabbit mAb (Pacific Blue™ Conjugate) (green) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control (Pacific Blue™ Conjugate) #9078 (red).

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Flow Cytometry, Methanol Permeabilization Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5-1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 15 min at room temperature.
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS.

C. Permeabilization

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

  1. Aliquot desired number of cells into tubes or wells.
  2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
  3. Resuspend cells in 100 µl of diluted antibody conjugate (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in incubation buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 5 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised June 2017

Protocol Id: 407

Product Usage Information

Application Dilutions
Flow Cytometry 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.

Specificity / Sensitivity

c-Myc (D84C12) Rabbit mAb (Pacific Blue™ Conjugate) detects endogenous levels of total c-Myc protein. This antibody is not recommended for detection of Myc-tagged fusion proteins. For detection of Myc-tagged fusion proteins use Myc-Tag (9B11) Mouse mAb #2276 or Myc-Tag (71D10) Rabbit mAb #2278.


Species Reactivity: Human, Mouse, Rat
Species predicted to react based on 100% sequence homology: Dog, Pig

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to amino-terminal residues of c-Myc protein.

Product Description

This Cell Signaling Technology antibody is conjugated to Pacific Blue™ fluorescent dye and tested in-house for direct flow cytometry in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated antibody c-Myc (D84C12) Rabbit mAb #5605.


Members of the Myc/Max/Mad network function as transcriptional regulators with roles in various aspects of cell behavior including proliferation, differentiation and apoptosis (1). These proteins share a common basic-helix-loop-helix leucine zipper (bHLH-ZIP) motif required for dimerization and DNA-binding. Max was originally discovered based on its ability to associate with c-Myc and found to be required for the ability of Myc to bind DNA and activate transcription (2). Subsequently, Max has been viewed as a central component of the transcriptional network, forming homodimers as well as heterodimers with other members of the Myc and Mad families (1). The association between Max and either Myc or Mad can have opposing effects on transcriptional regulation and cell behavior (1). The Mad family consists of four related proteins; Mad1, Mad2 (Mxi1), Mad3 and Mad4, and the more distantly related members of the bHLH-ZIP family, Mnt and Mga. Like Myc, the Mad proteins are tightly regulated with short half-lives. In general, Mad family members interfere with Myc-mediated processes such as proliferation, transformation and prevention of apoptosis by inhibiting transcription (3,4).


1.  Baudino, T.A. and Cleveland, J.L. (2001) Mol Cell Biol 21, 691-702.

2.  Blackwood, E.M. and Eisenman, R.N. (1991) Science 251, 1211-7.

3.  Henriksson, M. and Lüscher, B. (1996) Adv Cancer Res 68, 109-82.

4.  Grandori, C. et al. (2000) Annu Rev Cell Dev Biol 16, 653-99.


Entrez-Gene Id 4609
Swiss-Prot Acc. P01106


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Pacific Blue is a trademark of Molecular Probes, Inc.

14426
c-Myc (D84C12) Rabbit mAb (Pacific Blue™ Conjugate)