Invitrogen™ Phospho-IkB alpha (Ser32, Ser36) Monoclonal Antibody (RILYB3R), eFluor™ 660, eBioscience™, Invitrogen™

Catalog No.	Quantity
50-112-4487	100 Tests
50-112-4486	25 Tests

Catalog No. 50-112-4487 Supplier Invitrogen™ Supplier No. 50903542

Description

Mouse Monoclonal Antibody

Description: This RILYB3R monoclonal antibody recognizes human and mouse nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I kappa B alpha) when phosphorylated on serines 32 and 36 (S32/S36). I kappa B alpha is one member of a family of cellular proteins that functions to inhibit classical/canonical NF-kappa B signaling by masking the nuclear localization signals (NLS) of NF-kappa B proteins and keeping them sequestered in an inactive state in the cytoplasm. Classical/canonical NF-kappa B signaling is initiated in response to myriad stimuli including engagement of T cell and B cell receptors, growth factors, and inflammatory stimuli (reactive oxygen species, TNF alpha, IL-1) and results in the activation of the I kappa B kinase (IKK) complex that includes IKK alpha, IKK beta, and NEMO. IKK phosphorylates I kappa B alpha resulting in its ubiquitination, degradation, and subsequent translocation of NF-kappa B transcription factor proteins into the nucleus. Applications Reported: This RILYB3R antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This RILYB3R 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.

IkB-alpha is a 40 kDa protein that functions to inhibit NF- kappaB activity. The inhibition occurs via protein-protein interaction between I kappaB proteins and NF- kappaB dimers in the cytosol. The interaction of I kappa B-alpha with NF- kappaB masks the nuclear localization sequence of NF- kappaB, preventing NF- kappaB translocation to the nucleus. A variety of stimuli can activate gene expression by liberating NF- kappaB through the degradation of I kappaB alpha. These stimuli include the proinflammatory cytokines TNF- alpha and IL-1 beta, chemokines, PMA, growth factors, LPS, UV irradiation, viral infection, as well as various chemical and physical stresses. In humans, the gene is located on the q arm of chromosome 14. Activation of NFkB requires that IkB be phosphorylated on specific serine residues, which results in targeted degradation of IkB. IkB kinase alpha (IKK alpha), previously designated CHUK, interacts with IkB-alpha and specifically phosphorylates IkB-alpha on the sites that trigger its degradation Serines 32 and 36. IKK alpha appears to be critical for NFkB activation in response to proinflammatory cytokines. Phosphorylation of IkB by IKK alpha is stimulated by the NFkB inducing kinase (NIK), which itself is a central regulator for NFkB activation in response to TNF and IL-1. The functional IKK complex contains three subunits, IKK alpha, IKK beta and IKK gamma, and each appear to make essential contributions to IkB phosphorylation.

Specifications

• Antigen: Phospho-IkB alpha (Ser32, Ser36)
• Applications: Flow Cytometry
• Classification: Monoclonal
• Clone: RILYB3R
• Concentration: 5 μL/Test
• Conjugate: eFluor 660
• Formulation: PBS with BSA and 0.09% sodium azide; pH 7.2
• Gene: NFKBIA
• Gene Accession No.: P25963, Q9Z1E3
• Gene Alias: AI462015; ECI-6; ECI-6/IKBA; I kappa B-alpha; I(Kappa)B(alpha); I79_018146; IkappaB alpha; IkappaBalpha; I-kappaBalpha; I-kappa-B-alpha; Ikba; IKBalpha; IKB-alpha; Inhibitor of nuclear factor of kappa light chain gene enhancer in B-cells alpha; Inhibitor of nuclear factor of kappa light chain gene enhancer in B-cells, alpha; MAD3; MAD-3; Major histocompatibility complex enhancer-binding protein MAD3; NF kappa B inhibitor alpha; NF-kappaB inhibitor alpha; NF-kappa-B inhibitor alpha; NFKB inhibitor alpha; NFKBI; Nfkbia; nuclear factor of kappa light chain gene enhancer in B-cells; nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, alpha; nuclear factor of kappa light polyp gene enhancer in B-cell 1; nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha; nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha; Rel-associated pp40; REL-associated protein pp40; RL/IF-1
• Gene Symbols: NFKBIA
• Host Species: Mouse
• Purification Method: Affinity chromatography
• Quantity: 100 Tests
• Regulatory Status: RUO
• Primary or Secondary: Primary
• Gene ID (Entrez): 18035, 4792
• 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).

Is the eFluor 660 fluorochrome compatible with Anti-Cy5/Alexa Fluor 647 beads?

Yes, in-house studies have demonstrated that the eFluor 660 fluorochrome is recognized by Anti-Cy5/Alexa Fluor 647 beads. Side by side studies with Alexa Fluor 647 versus eFluor 660 conjugated antibodies have demonstrated comparable results.

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.

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.

Safety and Handling

WARNING: Reproductive Harm - www.P65Warnings.ca.gov

For Research Use Only.