Invitrogen™ Phospho-MCL-1 (Ser159) Monoclonal Antibody (RBCERNR), PE, eBioscience™

Catalog No.	Quantity
12-903-842	100 Tests

Catalog No. 12-903-842 Supplier Invitrogen™ Supplier No. 12903842

Description

Mouse Monoclonal Antibody

This RBCERNR monoclonal antibody recognizes human and mouse myeloid cell leukemia sequence 1 (Mcl-1) when phosphorylated on serine 159 (S159). Mcl-1 is an anti-apoptotic protein that is a member of the Bcl-2 family of proteins important for regulation of cell survival/apoptosis. Mcl-1 is primarily localized to the outer membrane of mitochondria where it prevents cytochrome c release via dimerization with other Bcl-2 family members such as Bim. PI3K activation of AKT results in the phosphorylation of GSK3 beta at serine 9 (S9) resulting in destabilization and degradation of GSK3 beta. Loss of GSK3 beta prevents phosphorylation of Mcl-1 on S159 and its subsequent ubiquitination and degradation. Mice conditionally lacking Mcl-1 in lymphocytes showed that Mcl-1 is essential during early lymphoid development and for the maintenance of mature lymphocytes. Applications Reported:This RBCERNR antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This RBCERNR antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 μL (0.125 μg) per test. A test is defined as the amount (μg) of antibody that will stain a cell sample in a final volume of 100 μL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Staining Protocol: Protocol A and Protocol C are recom...

MCL1 (Myeloid cell leukemia-1) belongs to the Bcl-2 family and is involved in programing, differentiation and concomitant maintenance of cell viability, but not of proliferation. Isoform 1 of MCL1 inhibits apoptosis while isoform 2 promotes it. The carboxy terminal of MCL1 and bcl-2 share significant sequence homology. Expression of MCL1 is increased upon exposure of ML-1 cells to various types of DNA damaging agents (e.g. ionizing radiation, ultraviolet radiation, and alkylating drugs) along with increases in GADD45 and Bax and a decrease in bcl-2. Enhanced expression of MCL1, prominently associated with mitochondria, complements the continued expression of bcl-2 in ML-1 cells undergoing differentiation. Like bcl-2, MCL1 has the capacity to promote cell viability under conditions that otherwise cause apoptosis. While the mechanism by which MCL1 inhibits apoptosis is not known, it is thought that it may heterodimerize and neutralize pro-apoptotic members of the Bcl-2 family such as Bim or Bak. MCL1 was originally identified in differentiating myeloid cells, but has since been shown to be expressed in multiple cell types. MCL1 is essential for embryogenesis and for the development and maintenance of B and T lymphocytes in animals. MCL1 exists as at least two distinct isoforms designated MCL1L and MCL1S. In marked contrast to the larger isoform of MCL1, overexpression of MCL1S promotes cell death.

Specifications

• Antigen: Phospho-MCL-1 (Ser159)
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: RBCERNR
• Concentration: 5 μL/Test
• Conjugate: PE
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: MCL1
• Gene Accession No.: P97287, Q07820
• Gene Alias: AW556805; BCL2 family apoptosis regulator; BCL2L3; bcl2-L-3; Bcl-2-like protein 3; bcl-2-related protein EAT/mcl1; EAT; Induced myeloid leukemia cell differentiation protein Mcl-1; induced myeloid leukemia cell differentiation protein Mcl-1 homolog; Mcl1; Mcl-1; mcl1/EAT; MCL1-ES; MCL1L; MCL1S; MCL-1S; MGC104264; MGC1839; myeloid cell leukemia 1; myeloid cell leukemia ES; myeloid cell leukemia sequence 1; myeloid cell leukemia sequence 1 (BCL2-related); TM
• Gene Symbols: MCL1
• Host Species: Mouse
• Purification Method: Affinity chromatography
• Quantity: 100 Tests
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 17210, 4170
• Target Species: Human, Mouse
• Content And Storage: 4°C, store in dark, DO NOT FREEZE!
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG2b κ

Frequently Asked Questions (FAQs)

How does eBioscience determine the specificity of their phosphorylation-specific antibodies?

Antibodies are tested and validated in a variety of ways. First, we verify that in a panel of cells in which different pathways have been induced, we see phosphorylation specific staining only in the cells in which the specific pathway of interest has been activated. Second, we verify that phosphorylation specific staining is observed only in cell types in which the protein is expressed and not in cell types in which the protein is not expressed. Third, whenever possible, western immunoblotting is used to confirm the presence of a band(s) of the appropriate size(s) in stimulated/treated cells (and not in unstimulated/untreated cells, as appropriate).

How long can cells that have been fixed and placed in methanol be stored for use with eBioscience antibodies? What is the recommended storage temperature? How about storage of fixed cells in eBioscience fixation buffers?

We have data demonstrating that fixed cells stored in methanol are stable at -20 degrees C or at -80 degrees C for several weeks. We do not recommend storing fixed cells in eBioscience Foxp3/Transcription Factor Buffer. However, cells fixed with eBioscience Foxp3/Transcription Factor Buffer or eBioscience Intracellular (IC) fixation buffer can be stored in eBioscience IC fixation buffer for up to 3 days at 4 degrees C in the dark.
Note: Please be aware that higher compensation values may be seen with tandem dyes if any other fixative is used apart from eBioscience IC fixation buffer, for 3-day storage.

What is the optimal protocol for eBioscience phosphorylation-specific antibodies?

Each antibody has been tested in three different buffer systems: IC fixation and permeabilization, Foxp3/Transcription Factor Buffer, and IC fixation Buffer/Methanol. The recommended buffer system(s) will be noted on the Technical Data Sheet for the specific antibody.

What is the best way to maintain surface staining when using the methanol-based protocol?

Because methanol can destroy the epitope recognized by some antibodies, ideally surface staining would be performed at the beginning of the protocol with a methanol-resistant fluorochrome (like eFluor 450, FITC, and eFluor 660).
Please recognize that surface staining before stimulation can have undesired effects due to activation of the cell caused by antibody binding. We recommend staining after stimulation/treatment, fixation, and methanol treatment.

Can I use other buffer systems from other companies when using eBioscience phosphorylation-specific antibodies?

In general, you can use other companies' buffer systems provided that their buffers are of a similar composition as the buffers recommended. Be aware that results will vary depending upon the buffer system used. eBioscience antibodies have been optimized for use in eBioscience buffers and we have not tested all of our antibodies in other buffer systems. Thus, we highly recommend using our optimized protocol and buffer systems.

What are the appropriate controls to run when using eBioscience phosphorylation-specific antibodies?

It is important to understand whether the stimulation/treatment results in an upregulation or a down regulation of the phosphorylation event. Thus, it is recommended that changes in phosphorylation levels in stimulated/treated versus unstimulated/untreated cells be compared. A histogram or density plot overlay these two treatments will provide a better assessment of a change in phosphorylation event than using isotype controls.

Can I perform phosphorylation specific staining and stain for intracellular cytokines at the same time when using eBioscience phosphorylation-specific antibodies?

In theory, if the buffer systems are compatible for the antibodies of interest, this can be done. In practice, cytokine translation and phosphorylation events do not typically occur in the same timeframe, therefore, it may be difficult to detect both events in the same sample. The results will be depended on the kinetics and should be determined for each system and protein of interest.

When using eBioscience phosphorylation-specific antibodies, can I perform phosphorylation specific staining together with transcription factor staining at the same time? If so, what buffer and protocol do I use?

In theory, this can be done if the antibodies against both the transcription factor and the phosphorylated protein work in the same buffer system. For example, Anti-Human/Mouse phsopho-H2AX and all transcription factor eBioscience antibodies offered will work in the Foxp3/Transcription Factor Buffer System (Cat. No. 00-5523-00).

For eBioscience phosphorylation-specific antibodies, can total and phosphorylated protein be analyzed in parallel?

In theory, this can be done if the antibodies to both proteins work in the same buffer system and if the antibodies recognize different epitopes. Please refer to the individual Technical Data Sheets for information about buffer compatibility. Also, consider using eBioscience InstantOne ELISA Kits for immunoassay quantitation of both total and phosphorylated proteins such as AKT1/2/3, JNK1/2/3, NFκB, STAT3, and STAT5.