Hot Summer. Cool Savings. | Start Saving >>
35876
c-Myc (D3N8F) Rabbit mAb (PE Conjugate)

c-Myc (D3N8F) Rabbit mAb (PE Conjugate) #35876

APPLICATIONS

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

Flow cytometric analysis of HT-29 cells using c-Myc (D3N8F) Rabbit mAb (PE Conjugate) (solid line) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line).

Learn more about how we get our images

Flow Cytometry Triton™ X-100 Permeabilization Protocol - Conjugated Antibody

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. Adjust pH to 8.0.
  2. Formaldehyde (methanol free).
  3. Incubation Buffer (1X PBS / 0.5% BSA): Dissolve 0.5 g bovine serum albumin (BSA) in 100 mL 1X PBS. Store at 4°C.
  4. Permeabilization Buffer (1X PBS / 0.3% Triton™ X-100 / 0.5% BSA): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml Incubation Buffer.

B. Fixation and Permabilization

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 briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
  3. Fix for 15 minutes at room temperature (20-25 °C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container.
  5. Resuspend cells in 1 ml Permeabilization Buffer.
  6. Incubate for 10 minutes at room temperature.
  7. Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.

C. Staining Using Conjugated Primary Antibody

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells (suggested range of 0.5x106 to 1x106 cells).
  2. Centrifuge cells and discard supernatant.
  3. Resuspend cells in 100 µl of diluted conjugated primary antibody (prepared in Incubation Buffer at the recommended dilution).
  4. Incubate for 30-60 min at room temperature (20-25 °C). Protect from light.
  5. Wash by centrifugation in 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section D).

D. Optional DNA Dye

  1. Resuspend cells in 0.1-0.5 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 January 2017

revised May 2018

Protocol Id: 1344

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.

c-Myc (D3N8F) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total c-Myc protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to a central region within human c-Myc protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometry analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated c-Myc (D3N8F) Rabbit mAb #13987.

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.
XP is a registered trademark of Cell Signaling Technology, Inc.

Upstream / Downstream

pathwayImage

Explore pathways related to this product.

To Purchase # 35876S

View sizes
Product # Size Price
35876S
100 µl (50 tests) $ 299.0

Related Products