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Pathway Focus: Metabolic Disorders

Measuring Acute Phase Proteins in Metabolic Research
Measuring Acute-Phase Proteins in Metabolic Research—Sandwich ELISA Kits
Accurate Detection of CholesterolAccurate Detection of Cholesterol in Your Research Samples—The Amplex® Red Cholesterol Assay Kit

Akt Kinase Signaling Cascade in Type 1 DiabetesAkt Kinase Signaling Cascade in Type 1 Diabetes—Antibodies for Metabolic Research
 

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Measuring Acute-Phase Proteins in Metabolic Research—Sandwich ELISA Kits

  • Quickly detect specific proteins
  • Easy-to-use assay
  • Reproducible results



Metabolic syndrome is a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes. Serum amyloid A (SAA), c-reactive protein (CRP), and plasminogen activator inhibitor-1 (PAI-1) play important roles in these diseases. SAA and CRP are acute-phase proteins with plasma concentrations that increase in response to inflammation; inflammatory cells secrete a variety of cytokines into the bloodstream, including IL-6 and TNFα.

The liver responds by producing a large number of these acute-phase reactants. One type of acute-phase protein is plasminogen, which functions to degrade blood clots (fibrinolysis). PAI-1 inhibits the fibrinolysis process.

We offer sandwich ELISA kits to quickly detect and quantify specific proteins to support your normal and diseased model research. The ELISA kits allow results to be collected in an easy and reproducible fashion. Calibrated standard curves are provided to help accurately quantify the level of protein in each experimental run. ELISA technology allows a more detailed understanding of protein levels in metabolic disorders.
Standard curve of Human SAA ELISA Kit
Figure 1. Standard curve for the Human SAA ELISA Kit.

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The dynamic range of the assay is 9.4–600 ng/mL with an analytical sensitivity of 4 ng/mL.
For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated.

Accurate Detection of Cholesterol in Your Research Samples—The Amplex® Red Cholesterol Assay Kit

  • Simple—detect cholesterol in research samples; no processing required
  • Versatile—use with fluorescence or colorimetric detection
  • Sensitive—detect as little 80 ng/mL (200 nM)



The Amplex® Red Cholesterol Assay Kit provides a very simple and sensitive fluorometric method to permit accurate quantitation of cholesterol in research samples using a fluorescence microplate reader or fluorometer. Because a large portion of cholesterol in blood is in the form of cholesteryl esters, the assay is based on an enzyme-coupled reaction that detects both free cholesterol and cholesteryl esters.
Cholesteryl esters are hydrolyzed by cholesterol esterase into cholesterol, which is then oxidized by cholesterol oxidase to yield H2O2 and the corresponding ketone product. The H2O2 is then detected using Amplex® Red reagent, a highly sensitive and stable probe for H2O2. In the presence of horseradish peroxidase (HRP), Amplex® Red reagent reacts with H2O2 in a 1:1 stoichiometry to produce highly fluorescent resorufin. Because resorufin has absorption/emission maxima of ~571/585 nm, there is little interference from autofluorescence in most biological samples.

Detection of cholesterol using the Amplex® Red Cholesterol Assay Kit

Figure 2. Detection of cholesterol using the Amplex® Red Cholesterol Assay Kit.

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Each reaction contained 150 µM Amplex® Red reagent, 1 U/mL HRP, 1 U/mL cholesterol oxidase, 1 U/mL cholesterol esterase, and the indicated amount of cholesterol in 1X reaction buffer. Reactions were incubated at 37°C for 30 min. Fluorescence was measured with a fluorescence microplate reader using excitation at 560 ± 10 nm and fluorescence detection at 590 ± 10 nm. The inset shows the high sensitivity and excellent linearity of the assay at low levels of cholesterol (0–0.015 µg/mL).
For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated.

Akt Kinase Signaling Cascade in Type 1 Diabetes—Antibodies for Metabolic Research

  • Measure total and phosphorylated proteins
  • Highly sensitive and specific antibodies
  • Excellent lot-to-lot consistency with ABfinity™ technology



Akt/protein kinase B (Akt) and its signaling cascade are involved in a number of different cellular functions, including glucose uptake, glycogen synthesis, cell growth, survival, apoptosis, protein synthesis, and endothelial nitric oxide production. In Type 1 diabetes, the kidney is exposed to fluctuating levels of blood glucose and exogenous insulin, which is administered as the major treatment of the disease. It has been well established that both glucose and insulin can modulate Akt activity in various cell types and tissues, and thus can affect the signaling cascade and subsequent cellular processes.
We offer pan and phosphorylation site–specific antibodies that can be used in research to check the activation status of Akt, mTOR, 4E-BP1, and other targets in the signaling cascade. ABfinity™ antibodies are rabbit recombinant antibodies produced by cloning the protein-specific genes and expressing them in mammalian cells. The result is a highly specific antibody with consistent lot-to-lot performance. New ABfinity™ antibodies include an antibody against Akt that is phosphorylated at serine 473, as well as antibodies against RANBP3,CD117 (c-Kit)[pY703], MEK2, SAA1, and Hexokinase 2, which are all involved in Akt or related pathways.

Western blot and peptide competition.
Figure 3. Western blot and peptide competition. Extracts of NIH3T3 cells unstimulated (lane 1) or stimulated with 50 ng/mL PDGF for 5 min (lanes 2–5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF.

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The membrane was blocked with a 5% BSA-TBST buffer for 1 hr at room temperature, then incubated with the Akt/PKB [pS473] rabbit monoclonal antibody for 2 hr at room temperature in a 1% BSA-TBST buffer, following prior incubation with: no peptide (lanes 1 and 2), the nonphosphorylated peptide 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. The data show that only the phosphopeptide corresponding to Akt/PKB [pS473] completely blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of Akt/PKB [pS473] phosphorylation by the addition of PDGF in this cell system.
ABfinity™ Recombinant Rabbit Monoclonal Antibody Selection Guide

Product Applications
 Clone Reactive Species
Cat No.
RANBP3 ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified
WB, ICC/IF
B24H15L41
Human
700076
CD117 (c-Kit)[pY703]ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified
WB
53H8L29
Human
700135
MEK2 ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified
WB, ICC/IF
B19H36L8
Human
700829
SAA1 ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified
WB
D9H4L41
Mouse
700830
Hexokinase 2 ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified
WB
2H8L6
Human
700422
AKT [pS473] ABfinity™ Recombinant Rabbit Monoclonal Antibody - Purified
ELISA
99H9L9
Human
44621G

ICC = immunocytochemistry. IF = immunofluorescence. WB = western blot.
For research use only. Not intended for any animal or human therapeutic or diagnostic use, unless otherwise stated.