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55673
Numb (C29G11) Rabbit mAb (PE Conjugate)

Numb (C29G11) Rabbit mAb (PE Conjugate) #55673

APPLICATIONS

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

Flow cytometric analysis of HeLa cells using Numb (C29G11) Rabbit mAb (PE Conjugate) (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (PE Conjugate) #5742 (dashed line).

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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

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.

Numb (C29G11) Rabbit mAb (PE Conjugate) detects endogenous levels of total Numb protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala570 of human Numb protein.

This Cell Signaling Technology antibody is conjugated to phycoerythrin (PE) and tested in-house for direct flow cytometric analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated Numb (C29G11) Rabbit mAb #2756.

Numb contains an amino-terminal phosphotyrosine-binding (PTB) domain and carboxy-terminal endocytic binding motifs for α-adaptin and EH (Eps15 homology) domain-containing proteins, indicating a role in endocytosis (1,2). There are four mammalian Numb splicing isoforms that are differentially expressed and may have distinct functions (3-5). Numb acts as a negative regulator of Notch signaling by promoting ubiquitination and degradation of Notch (6). The protein is asymmetrically segregated into one daughter cell during cell division, producing two daughter cells with different responses to Notch signaling and different cell fates (7,8). The localization of Numb can also be regulated by G-protein coupled receptor (GPCR) and PKC signaling (9).

  1. Berdnik, D. et al. (2002) Dev. Cell 3, 221-231.
  2. Santolini, E. et al. (2000) J. Cell Biol. 151, 1345-1352.
  3. Dho, S.E. et al. (1999) J. Biol. Chem. 274, 33097-33104.
  4. Verdi, J.M. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 10472-10476.
  5. Toriya, M. et al. (2006) Dev. Neurosci. 28, 142-155.
  6. McGill, M.A. and McGlade, C.J. (2003) J. Biol. Chem. 278, 23196-23203.
  7. Verdi, J.M. et al. (1996) Curr. Biol. 6, 1134-1145.
  8. Reugels, A.M. et al. (2006) Dev. Dyn. 235, 934-948.
  9. Dho, S.E. et al. (2006) Mol. Biol. Cell 17, 4142-4155.
Entrez-Gene Id
8650
Swiss-Prot Acc.
P49757
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.

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To Purchase # 55673S

Product Number Size Price
55673S 100 µl (50 tests) $299.00.0
Quantity Subtotal
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