BioPath Online

NFкB and IкBα Protein Measurement Using PhosphoELISA™ Kits

  • Quickly detect and quantify transcription factors
  • Reliable results illuminate role of NFκB liberation
  • Better understand role in immune disorders and inflammation

Nuclear factor-kappa B (NFκB) transcription factors are a group of protein dimers, which comprise various combinations of members of the NFκB/Rel protein family. Through its regulation of NFκB, the inhibitor of nuclear factor-κB α isoform (IκBα) controls immune and inflammatory responses, cell division, and apoptosis.

NFκB mediates inflammatory responses, immune responses, responses to viral infections, cell division, and regulation of apoptosis, serving both as an anti-apoptotic and pro-apoptotic signal. Disease states in which NFκB is improperly regulated include cancer, neurodegenerative diseases, arthritis, asthma, and inflammatory bowel disease.

Underscoring the importance of regulation of NFκB by IκB in cancer is the observation that many multiple myelomas possess polymorphisms at IκB regulatory sites leading to improper regulation of NFκB signaling. 

A variety of stimuli activate gene expression by liberating NFκB through degradation of IκBα:

  • Proinflammatory cytokines TNFα and IL-1β
  • Chemokines
  • PMA
  • Growth factors
  • LPS
  • UV irradiation
  • Viral infection
  • Chemical and physical stresses

The events leading to this liberation are well-defined. In response to stimulus, serine residues 32 and 36 of IκBα are phosphorylated; this serine phosphorylation provides a signal for IκBα E3 ligase, a protein complex composed of SKP-1, Cul-1, Roc1, and Fbw1.

Invitrogen™ sandwich ELISA kits quickly detect and quantify transcription factors in cell lysate models (Figure 1). The ELISA kits allow high throughput of samples in a 96-well format and reproducible, quantitative results. Calibrated standard curves quantify the level of protein in each experimental run. The ELISA technology allows a more detailed understanding of proteins in specific NFкB pathways (Figure 2).
Jurkat cells were treated with TNF-a

Figure 1. Jurkat cells (5 x 106) treated with TNFα at 20 ng/mL for 15 and 30 min. The cells were harvested, resuspended in hypotonic lysis buffer, and centrifuged to isolate nuclei. The nuclei were lysed with extraction buffer and resulting lysates tested by ELISA and Western blot. The data show that the NFκBp65 total ELISA kit can monitor and quantitate p65 translocation into the nucleus, consistent with western blot analysis (insert).

IkBa phosphorylation was observed 2 minutes after TNF-a treatment

Figure 2. IκBα phosphorylation was observed 2 min after TNFα treatment followed by a rapid decrease due to its subsequent degradation. The results correlate well with western blot analyses of the same samples (inset in the graph below).

Measuring Insulin and its Receptors Using ELISA

  • Highly specific antibodies validated against a variety of species
  • More than 300 phospho site-specific antibodies
  • Antibodies recognize NFκBs, RelB, TRAF6, IκBα, AKT, TLC, and FAK

To help researchers in their NFκB signal transduction research, Invitrogen offers highly specific antibodies. These antibodies are validated against a variety of species and with most applications.  Among these are phosphor site–specific antibodies, which only detect phosphorylated proteins. To make sure these antibodies detect only phosphorylated proteins, each lot is validated with peptide competition experiments (Figure 3).

Invitrogen offers over 300 phospho site–specific antibodies to study the NFκB and related pathways. These antibodies include a variety of antibodies recognizing NFκB, RelB, TRAF6, IκBα, and related pathways such as AKT, TLC, and FAK.

Figure 3. Demonstration of antibody specificity. Extracts of Jurkat cells unstimulated (lane 1) or stimulated with 100 ng/mL PMA for 20 min then 0.5 μM Ca2+ ionophore for 10 additional minutes (lanes 2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% nonfat dried milk–TBST buffer, then incubated with the NFκB [pS529] antibody for two hours at room temperature in a 3% nonfat dried milk–TBST buffer, following prior incubation with: no peptide (lanes 1, 2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (lane 3), a generic phosphoserine-containing peptide (lane 4), or the phosphopeptide immunogen (lane 5). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignal™ method. Only the phosphopeptide corresponding to NFκB [pS529] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show induction of the phosphorylated signal after addition of PMA and a Ca2+ ionophore.
Extracts of Jurkat cells unstimulated or stimulated with 100 ng/mL PMA for 20 minutes

Recombinant TNFα for NFκB Pathway Research

  • Explore the link between TNFα and immunity/inflammation
  • Multiple species of GIBCO® bioactive cytokine
  • Convenient packages for in vitro study

Tumor necrosis factor alpha (TNFα) is a key early response mediator involved in many aspects of innate and acquired immunity. Overproduction of this cytokine is associated with diseases such as rheumatoid arthritis. TNFα was originally described as cachectin, a protein involved in the development of cachexia. Later studies described the proatherogenic changes in lipid metabolism induced by this cytokine. In animal models, administration of TNFα led to severe impairment of glucose tolerance and insulin sensitivity. Thus, TNFα might serve as a therapeutic target in these disorders. Treatment of sepsis with agents that block TNFα actions worsened these conditions, whereas in cases of chronic inflammation, such as rheumatoid arthritis, this therapy was highly beneficial.
The NFκB transcription factor family consists of five members—p50, p52, p65 (RelA), c-Rel, and RelB—which can form various homo- and heterodimers. NFκB is normally sequestered in the cytoplasm by proteins of the IκB family; IκBα, β, and γ. The predominant induced form of NFκB, a p50 and p65 heterodimer, translocates to the nucleus upon activation. Common activating stimuli of the NFκB pathway include interleukin-1 (IL-1), TNFα, lipopolysaccharide (LPS), hypoxia/reoxygenation, and phorbol myristate acetate (PMA).

Invitrogen offers multiple species of GIBCO® biologically active TNFα in convenient package sizes for in vitro studies of TNF mediation of inflammatory processes.