Invitrogen™ Phospho-AKT1 (Ser473) Monoclonal Antibody (SDRNR), eFluor™ 450, eBioscience™

Catalog No.	Quantity
48-971-542	100 Tests

Catalog No. 48-971-542 Supplier Invitrogen™ Supplier No. 48971542

Description

Mouse Monoclonal Antibody

Description: This SDRNR monoclonal antibody recognizes human and mouse AKT (also known as Protein Kinase B (PKB)) when phosphorylated on S473. AKT is a serine/threonine protein kinase that plays a key role in multiple cellular processes including metabolism, proliferation, apoptosis/survival, and migration. There are three homologous isoforms of AKT: AKT1, AKT2, and AKT3. AKT is activated by binding of its pleckstrin homology (PH) domain to membrane phospholipids and by phosphorylation. Phosphorylation of AKT at T308 by PDK1 and at S473 is required for full activation of this kinase. AKT promotes cell survival by inhibiting apoptosis via phosphorylation and inactivation of several targets including Bad, Foxo1, c-Raf, and caspase-9. Deregulation of AKT has been implicated as a major contributing factor in many types of cancer. AKT is negatively regulated by the phosphatase PTEN as well as by the chemical inhibitor LY294002. Specificity of this SDRNR clone was determined by ELISA, flow cytometry, and western blotting. Applications Reported:This SDRNR antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This SDRNR antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of normal human peripheral blood mononuclear cells. This can be used at 5 μL (0.5 μg) per test. A test is defined as the amount (μg) of antibody that will stain a cell sample in a final v...

AKT1 (PKB alpha) is a serine/threonine kinase that regulates cell survival. The activated enzyme inhibits apoptosis and stimulates cell cycle progression by phosphorylating numerous targets in various cell types, including cancer cells. This protein kinase is activated by insulin, PI3K, IGF1 and various other growth and survival factors. Akt promotes cell survival by inhibiting apoptosis through phosphorylation and inactivation of several targets, including forkhead transcription factors, and caspase-9. The AKT pathway is a major target for cancer drug discovery.

Specifications

• Antigen: Phospho-AKT1 (Ser473)
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: SDRNR
• Concentration: 5 μL/Test
• Conjugate: eFluor 450
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: AKT1
• Gene Accession No.: P31749, P31750
• Gene Alias: AKT; Akt kinase; AKT serine/threonine kinase 1; Akt/PKB; akt1; AKT1 kinase; AKT1m; Akt1m protein; Akt1-PA; Akt1-PB; Akt1-PC; Akt1-PD; Akt1-PE; CAKT; C-AKT; CG4006; CG4006-PA; CG4006-PB; CG4006-PC; CG4006-PD; CG4006-PE; CWS6; dAkt; D-Akt; dAkt kinase; dAKT/dPKB; dAkt/PKB; DAKT1; DAKT1/PKB; Dmel\CG4006; Dmel_CG4006; dPKB; DRAC-PK; DRAC-PK66; DRAC-PK85; l(3)04226; l(3)89Bq; murine thymoma viral (v-akt) oncogene homolog 1; pAkt; P-AKT; PKB; PKB alpha; PKB beta; PKB gamma; PKB/AKT; PKB/dAKT; PKBalpha; PKB-ALPHA; PKBG; PRKBA; protein kinase B; Protein kinase B alpha; protein kinase B-alpha; proto-oncogene c-Akt; RAC; rac protein kinase alpha; RAC protein kinase alpha RAC-PK alpha; RAC serine/threonine protein kinase; RAC serine/threonine-protein kinase; RAC-ALPHA; RAC-ALPHA SERINE/THREONINE-PROTEIN KINASE; RacPK; RAC-PK-alpha; RAC-PK-beta; RAC-PK-gamma; related to A and C kinases; related to PKA to PKC protein kinases; related to the A and C kinases; SD10374p; serine/threonine protein kinase; serine-threonine protein kinase; Thymoma viral proto-oncogene; thymoma viral proto-oncogene 1; v-akt murine thymoma viral oncogene homolog 1; V-AKT MURINE THYMOMA VIRAL ONCOGENE-LIKE PROTEIN 1
• Gene Symbols: AKT1
• Host Species: Mouse
• Purification Method: Affinity chromatography
• Quantity: 100 Tests
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 11651, 207
• Target Species: Human, Mouse
• Content And Storage: 4°C, store in dark, DO NOT FREEZE!
• Product Type: Antibody
• Form: Liquid
• Isotype: IgG2a κ

Frequently Asked Questions (FAQs)

I am unable to analyze my cells stained with eFluor Organic Dyes today. What options do I have?

Our options will depend on the samples you are analyzing.

If cell viability is not critical, you can store your stained samples at 4 degrees C or on ice overnight in the dark and analyze the following day.

For samples stained with eFluor organic fluorochromes, we recommend that cells be suspended in 100 uL of Flow Cytometry Staining Buffer (Cat. No. 00-4222) and 100 uL of eBioscience IC Fixation Buffer (Cat. No. 00-8222); samples can be incubated for up to 3 days at 4 degrees C in the dark. Alternatively, the 1-step Fix/Lyse Solution (Cat. No. 00-5333) can be used. This is a great option when working with whole blood but also works for other cell types.

Can the eFluor Organic fluorochromes be used for intracellular staining?

Yes, the eFluor Organic fluorochromes can be used for intracellular staining. The eFluor organic fluorochromes maintain bright signal and require minimal changes in compensation when fixed with eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) and Permeabilization Buffer (Cat. No. 00-8333-56) or 1-step Fix/Lyse Solution (Cat. No. 00-5333-54, 00-5333-57) (as compared to live cells).

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).

What buffers are compatible with the eFluor Organic fluorochromes and eVolve QDots?

The eFluor Organic fluorochromes and eVolve QDots can be used with flow staining buffers containing PBS and protein.

What is the difference between eFluor Organic Dyes and eVolve Qdots?

The eFluor Organic Dyes (eFluor 450, APC-eFluor 780, PerCP-eFluor 710, eFluor 710) are conventional fluorochromes. In contrast, the eVolve line of products are Quantum dots.

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.

Are the eFluor Organic Dyes photo-labile?

As with other fluorochromes, we recommend minimal exposure to light to maintain optimal signal.