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To Purchase # 65617S

65617S 100 µl (50 tests) $299.00.0
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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 L-929 cells using Mcl-1 (D2W9E) Rabbit mAb (PE Conjugate) (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #5742 (red).

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Flow Cytometry

Flow cytometric analysis of SK-OV-3 cells (blue) and MCF7 cells (green) using Mcl-1 (D2W9E) Rabbit mAb (PE Conjugate).

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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

Mcl-1 (D2W9E) Rabbit mAb (PE Conjugate) recognizes endogenous levels of total Mcl-1 protein.


Species Reactivity: Human, Mouse, Rat

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro60 of mouse Mcl-1 protein.

Product Description

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 Mcl-1 (D2W9E) Rabbit mAb #94296.


Mcl-1 is an anti-apoptotic member of the Bcl-2 family originally isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway (1). Similar to other Bcl-2 family members, Mcl-1 localizes to the mitochondria (2), interacts with and antagonizes pro-apoptotic Bcl-2 family members (3), and inhibits apoptosis induced by a number of cytotoxic stimuli (4). Mcl-1 differs from its other family members in its regulation at both the transcriptional and post-translational level. First, Mcl-1 has an extended amino-terminal PEST region, which is responsible for its relatively short half-life (1,2). Second, unlike other family members, Mcl-1 is rapidly transcribed via a PI3K/Akt dependent pathway, resulting in its increased expression during myeloid differentiation and cytokine stimulation (1,5-7). Mcl-1 is phosphorylated in response to treatment with phorbol ester, microtubule-damaging agents, oxidative stress, and cytokine withdrawal (8-11). Phosphorylation at Thr163, the conserved MAP kinase/ERK site located within the PEST region, slows Mcl-1 protein turnover (10) but may prime the GSK-3 mediated phosphorylation at Ser159 that leads to Mcl-1 destabilization (11). Mcl-1 deficiency in mice results in peri-implantation lethality (12). In addition, conditional disruption of the corresponding mcl-1 gene shows that Mcl-1 plays an important role in early lymphoid development and in the maintenance of mature lymphocytes (13).


1.  Kozopas, K.M. et al. (1993) Proc Natl Acad Sci USA 90, 3516-20.

2.  Yang, T. et al. (1995) J Cell Biol 128, 1173-84.

3.  Sato, T. et al. (1994) Proc Natl Acad Sci USA 91, 9238-42.

4.  Zhou, P. et al. (1997) Blood 89, 630-43.

5.  Wang, J.M. et al. (1999) Mol Cell Biol 19, 6195-206.

6.  Jourdan, M. et al. (2003) Oncogene 22, 2950-9.

7.  Chao, J.R. et al. (1998) Mol Cell Biol 18, 4883-98.

8.  Domina, A.M. et al. (2000) J Biol Chem 275, 21688-94.

9.  Inoshita, S. et al. (2002) J Biol Chem 277, 43730-4.

10.  Domina, A.M. et al. (2004) Oncogene 23, 5301-15.

11.  Maurer, U. et al. (2006) Mol Cell 21, 749-60.

12.  Rinkenberger, J.L. et al. (2000) Genes Dev 14, 23-7.

13.  Opferman, J.T. et al. (2003) Nature 426, 671-6.


Entrez-Gene Id 17210
Swiss-Prot Acc. P97287


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.

65617
Mcl-1 (D2W9E) Rabbit mAb (PE Conjugate)