Invitrogen™ Phospho-p38 MAPK (Thr180, Tyr182) Monoclonal Antibody (4NIT4KK), PerCP-eFluor™ 710, eBioscience™, Invitrogen™

Catalog No.	Quantity
46-907-842	100 Tests
46-907-841	25 Tests

Catalog No. 46-907-842 Supplier Invitrogen™ Supplier No. 46907842

Description

Mouse Monoclonal Antibody

Description: This 4NIT4KK monoclonal antibody recognizes human and mouse p38 mitogen-activated protein kinase (MAPK) when phosphorylated on T180/Y182. p38 MAPK belongs to a family of conserved serine/threonine protein kinases that are phosphorylated and activated in response to numerous stress stimuli including TLR ligands (such as LPS), osmotic shock, heat shock, UV irradiation, and inflammatory cytokines. There are four p38 MAPK members that include p38 alpha, p38 beta, p38 gamma, and p38 delta. The primary activators of p38 MAPK are MKK3/4 and MKK6. Several downstream targets of p38 MAPK have been identified including MK2/3, p53, ATF-2, and ETS1. p38 MAPK can be negatively regulated by the chemical inhibitor SB203580. Specificity of this 4NIT4KK clone was determined by ELISA, flow cytometry, and western blotting. Applications Reported: This 4NIT4KK antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This 4NIT4KK antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of stimulated normal human peripheral blood cells. This can be used at 5 μL (0.03 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.

The p38 MAPK cascade regulates a variety of cellular responses to stress, inflammation, and other signals. p38 MAPK is relatively inactive in the nonphosphorylated form and becomes rapidly activated by dual phosphorylation of a Thr-Gly-Tyr motifs. There are four isoforms of p38 MAPK, which differ in their tissue expression and affinity for upstream activators and downstream effectors. When cells are exposed to tumor necrosis factor, interleukin-1, heat shock, or other activating stimuli, activation of MAPK kinase-3 occurs by phosphorylation. Activated MAPK kinase-3/6 phosphorylate each residue of Thr180 and Tyr182 in p38 MAPK. Phospho-p38 MAPK activates ATF-2, CHOP-1, MEF-2 and other transcription factors through phosphorylation.

Specifications

• Antigen: Phospho-p38 MAPK (Thr180, Tyr182)
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: 4NIT4KK
• Concentration: 5 μL/Test
• Conjugate: PerCP-eFluor 710
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: MAPK14
• Gene Accession No.: P47811, Q16539
• Gene Alias: Crk1; CSAID-binding protein; Csaids binding protein; CSBP; CSBP1; CSBP2; CSPB1; cytokine suppressive anti-inflammatory drug binding protein; cytokine suppressive anti-inflammatory drug binding protein 1; cytokine suppressive anti-inflammatory drug-binding protein; cytokine-supressive anti-inflammatory drug binding protein; Exip; Hog; MAP kinase 11; MAP kinase 13; MAP kinase 14; MAP kinase 2; MAP kinase Mxi2; MAP kinase p38 alpha; MAP kinase p38 beta; MAP kinase p38 delta; MAPK 11; MAPK 13; MAPK 14; Mapk11; Mapk13; MAPK-13; Mapk14; MAX-interacting protein 2; mitogen activated protein kinase 11; mitogen activated protein kinase 13; mitogen activated protein kinase 14; mitogen-activated protein kinase 11; Mitogen-activated protein kinase 13; mitogen-activated protein kinase 14; mitogen-activated protein kinase 14A; mitogen-activated protein kinase p38 alpha; Mitogen-activated protein kinase p38 beta; mitogen-activated protein kinase p38 delta; mitogen-activated protein kinase p38-2; MXI2; p38; p38 alpha; p38 delta MAP kinase; p38 MAP kinase; p38 MAP kinase alpha; p38 MAPK; p38 mitogen activated protein kinase; p38-2; p38a; p38ALPHA; p38-alpha; p38alpha Exip; P38b; p38Beta; p38beta2; p38delta; p38Hog; p38MAPK; Prkm11; PRKM13; PRKM14; Prkm15; protein kinase, mitogen activated kinase, 11, p38beta; reactive kinase; RK; SAPK/Erk/kinase 4; SAPK2; SAPK2A; SAPK2B; SAPK4; Serk4; Stress-activated protein kinase 2a; Stress-activated protein kinase 2b; Stress-activated protein kinase 4; stress-activated protein kinase-2; stress-activated protein kinase-2b; tRNA synthetase cofactor p38
• Gene Symbols: MAPK14
• Host Species: Mouse
• Purification Method: Affinity chromatography
• Quantity: 100 Tests
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 1432, 26416
• 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)

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.

For Research Use Only.