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New Antibodies to Check the Balance Between Survival and Apoptosis in the Ras/Raf/MEK/ERK Signaling Cascade

  • ABfinity™ phospho-ERK/2 (T185/Y187) and MEK2 rabbit recombinant antibodies for sensitive detection
  • Designed for consistent performance for your assays
  • Consistent product from lot to lot


ABfinity™ recombinant rabbit monoclonal antibodies are produced from specific recombinant clones, so antibody performance is consistent over time. ABfinity™ antibodies are available against many targets, including ERK and MEK.

 

Recent studies have shown that malignant cells can hijack physiological pathways to promote their growth.

The Ras/Raf/MEK/ERK pathway is critical in regulating a range of biological functions, including cell differentiation, proliferation, senescence, cell survival, and apoptosis. Some estimates suggest that some aspect of this pathway is mutated in 30% of all cancers. Antibodies can help elucidate the balance between expression of these signaling proteins and cell fate. ABfinity™ recombinant monoclonal antibodies are ideal reagents for signaling pathway research. The ABfinity™ technology helps ensure consistent antibody performance so you don’t have to reoptimize your assay with each new lot.

Western blot of Jurkat lysates labeled with rabbit anti-ERK1/2 [pT185/pY187]

Figure 1. Western blot of Jurkat lysates labeled with rabbit anti-ERK1/2 [pT185/pY187].
Rabbit anti-ERK1/2 [pT185/pY187] (1 µg/mL) was used to label ERK1/2 in unstimulated Jurkat lysates (lane 1) or PMA-stimulated Jurkat lysates (lane 2).
For research use only. Not intended for any animal or human therapeutic or diagnostic use.


Tali™ Image-Based Cytometer and Tali™ Apoptosis Kit

  • Simultaneously stain apoptotic and necrotic cells in the sample
  • Visualize and quantify populations
  • Results comparable to flow cytometry analysis


The Tali™ Apoptosis Kit contains two fluorescent dyes: green-fluorescent Alexa Fluor® 488 dye conjugated to annexin V (an indicator of apoptosis), and red-fluorescent propidium iodide (an indicator of necrotic cells).

 

Use of these reagents with the Tali™ Image Cytometer enables the

 identification of apoptotic cells and the discrimination of apoptotic from necrotic cells in a population.

After you add the reagents to a sample and analyze it on the Tali™ Image Cytometer, three populations are revealed: apoptotic (green-fluorescent), dead (red- or yellow-fluorescent), and live (nonfluorescent).

Using the Tali™ Image-Based Cytometer and the Tali™ Apoptosis Kit, you can get rapid and quantitative apoptotic cell data.

Example of a Tali™ apoptosis assay using the Tali™ Apoptosis Assay Kit

Figure 2. Example of a Tali™ apoptosis assay using the Tali™ Apoptosis Assay Kit – Annexin V Alexa Fluor® 488 and Propidium Iodide.
Jurkat cells were induced with 10 µM camptothecin; at selected time points the Tali™ apoptosis assay was performed and the cells were analyzed with the Tali™ Image-Based Cytometer. The Sample tab shows the concentrations, relative proportions, and numbers of live, dead, and apoptotic cells. The image window (on the left) displays the captured fields of view; green-fluorescent apoptotic cells are clearly distinguishable from red-fluorescent dead cells and nonfluorescent live cells. Yellow-fluorescent cells are stained with both dyes and are recorded as dead.
For research use only. Not intended for any animal or human therapeutic or diagnostic use.


Second-Messenger Cyclic AMP Modulates the Apoptotic Program

  • Broad dynamic range—0.06 to 6,000 pmol (4–5 logs)
  • Flexible—can be used in manual or automated, high-throughput formats
  • Simple—sample dilution not required


Cyclic adenosine monophosphate (cAMP) is an important second messenger in many signal transduction pathways linking activation of cell-surface membrane receptors to intracellular responses and, ultimately, to changes in gene expression.

 

Typically after eating, insulin is released in response to rising levels of blood glucose. This competitive heterogeneous immunoassay measures cAMP directly from cell lysates at levels as low as 60 fmol to 6 µmol, over 4–5 orders of magnitude, without requiring any sample dilution. Detection is complete within 90 minutes, utilizes chemiluminescence, and is amenable to use in automated, high-throughput screening applications.

cAMP is known to modulate the apoptotic pathway in a wide variety of cells, accelerating apoptosis in some and delaying the rate of apoptosis in others (BMC Cancer 11:301 (2011)). 

The sensitivity and simplicity of the cAMP-Screen Direct® assay will enable researchers to measure small changes in levels of cAMP as they attempt to elucidate its function in apoptosis.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.


TNF Superfamily Proteins for Apoptosis Research

  • High biological activity
  • High purity
  • Proven compatibility with Gibco® media


Gibco® recombinant growth factors are highly purified, bioactive proteins. Gibco® cytokines and growth factors have been bioassayed with Gibco® media. Get your cytokines and growth factors from the media company you use and trust.

 

The TNF superfamily is perhaps the most widely studied group of apoptotic factors and is important to development and the immune system. Many of these proteins are transmembrane proteins that are cleaved to form soluble cytokines.

Life Technologies offers TNF-α, TRAIL, and other proteins involved in apoptosis. Gibco® cytokines and growth factors are high-purity recombinant proteins with high bioactivity. 

To help ensure that Gibco® growth factors are of the highest quality, each protein is analyzed for purity along with structural homogeneity so that a biologically active protein is produced.

For research use only. Not intended for any animal or human therapeutic or diagnostic use.


TNF-α Induces Anti-apoptotic Effects via the NF-κB Signaling Cascade

  • More sensitive than western blots—measure low-expressing proteins
  • Small volume of cell lysate needed—helps save precious samples
  • Quantitative results—no need to do densitometry analysis


NF-κB (nuclear factors of the kappa light polypeptide in B cells) proteins mediate inflammatory responses and cell division, and regulates apoptosis, serving both as an anti-apoptotic signal and as a pro-apoptotic signal. There are at least 5 members of the NF-κB protein family, including NF-κB1 (p50 and its precursor, p105), NF-κB2 (p52 and its precursor, p100), c-Rel, RelA (p65), and RelB. Dimers containing the p65 subunit serve as potent activators of transcription. Among mammalian NF-κB proteins the p65/p50 dimer is the most abundant. IκBα (inhibitor of nuclear factor-κB α isoform) masks the nuclear localization sequence of NF-κB in the cytoplasm, preventing translocation of NF-κB to the nucleus.

Diverse stimuli that activate NF-κB proteins through degradation of IκB include TNFα, IL-1B, viral infections, and various stresses.

 

Under resting conditions, NF-κB family members are sequestered in the cytoplasm in an inactive state through interaction with the IκB family members.

Phosphorylation of IκBα at serines 32 and 36 leads to its ubiquitination and degradation within the 26S proteasome. Degradation of IκBα liberates NF-κB transcription factors, allowing their rapid translocation from the cytoplasm to the nucleus, where they trigger transcription of the target genes. NF-κB can mediate upregulation of pro-apoptotic genes such as TRAIL, Fas, and Fas ligand.

The NF-κB p65 (Total) phosphoELISA™ Kit is designed to detect and quantify the level of NF-κB p65 protein, independent of its phosphorylation state. The IκBα [pS32] phosphoELISA™ Kit allows measurement of phosphorylated IkB. The IκBα (Total) phosphoELISA™ Kit is also available to normalize the amount of IκBα protein in your sample. ELISA is a more sensitive method than western blotting, allowing more accurate quantification of low-expressing proteins and also requiring smaller sample volumes per assay. Results are read on a microplate reader, so quantitative values are obtained in seconds.

  • Learn More About the Invitrogen™ ELISA Kits
Nuclei isolated from Jurkat cells

Figure 3. Nuclei isolated from Jurkat cells (treated with 20 ng/mL TNF-α) were analyzed with the NF-κB phosphoELISA™ Kit.
The data show that the NF-κB p65 (Total) phosphoELISA™ Kit can monitor and quantitate p65 translocation into the nucleus, which is consistent with western blot analysis.
For Research Use Only.  Not intended for any animal or human therapeutic or diagnostic use. © 2011 Life Technologies Corporation. All rights reserved.  The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.