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58326
Mcl-1 (D2W9E) Rabbit mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates

Mcl-1 (D2W9E) Rabbit mAb (Alexa Fluor® 488 Conjugate) #58326

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Flow cytometric analysis of L-929 cells using Mcl-1 (D2W9E) Rabbit mAb (Alexa Fluor® 488 Conjugate) (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 488 Conjugate) #2975 (red).

Flow cytometric analysis of SK-OV-3 cells (blue) and MCF7 cells (green) using Mcl-1 (D2W9E) Rabbit mAb (Alexa Fluor® 488 Conjugate).

Flow cytometric analysis of SK-OV-3 cells (blue) and MCF7 cells (green) using Mcl-1 (D2W9E) Rabbit mAb (Alexa Fluor® 488 Conjugate).

To Purchase # 58326S
Product # Size Price
58326S
100 µl  (50 tests) $ 305

Supporting Data

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

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye 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 Mcl-1 (D2W9E) Rabbit mAb #94296.

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.

Protocol

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Flow Cytometry, Methanol Permeabilization Protocol for Directly Conjugated Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
  2. 4% Formaldehyde, Methanol-Free (#47746)
  3. 100% Methanol (#13604): Chill before use
  4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com/flowdyes for a listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

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

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
    1. Alternatively, cells may be stored overnight at 4°C in 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. Permeabilize for a minimum of 10 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  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 primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
  4. Incubate for 1 hr at room temperature. Protect from light.
  5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted July 2009

revised August 2019

Protocol Id: 407

Specificity / Sensitivity

Mcl-1 (D2W9E) Rabbit mAb (Alexa Fluor® 488 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.

Background

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.

Pathways & Proteins

Explore pathways + proteins related to this product.

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.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
The Alexa Fluor dye conjugates in this product are sold under license from Life Technologies Corporation, for research use only excluding use in combination with DNA microarrays and high content screening (HCS).