Skip to Content
MilliporeSigma
All Photos(1)

Key Documents

View All Documentation

48-623MAG

Millipore

MILLIPLEX® Phospho/Total STAT3 Magnetic Bead 2-Plex Kit - Cell Signaling Multiplex Assay

Synonym(s):

2-Plex Phospho/Total Kit, Cell Signaling STAT3 Kit, STAT3 Bead Kit

Sign Into View Organizational & Contract Pricing
All Photos(1)

About This Item

UNSPSC Code:
12161503
NACRES:
NA.47
Pricing and availability is not currently available.

species reactivity

human, mouse

Quality Level

200

manufacturer/tradename

Milliplex®

technique(s)

multiplexing: suitable

detection method

fluorometric (Luminex® xMAP®)

storage temp.

2-8°C

General description

STATs (Signal Transducers and Activators of Transcription) are used by receptors for a wide variety of ligands including cytokines, hormones, growth factors and neurotransmitters. The phosphorylation of STAT proteins at conserved tyrosine residues activates SH2-mediated dimerization followed rapidly by nuclear translocation. STAT dimers bind to IRE (interferon response element) and GAS (gamma interferon-activated sequence) DNA elements, resulting in the transcriptional regulation of downstream genes. STAT pathways play important roles in oncogenesis, tumor progression, angiogenesis, cell motility, immune responses and stem cell differentiation.Protein phosphorylation represents the major mechanism used in regulating cellular functions in all eukaryotic cells. Aberrant phosphorylation has been implicated in the onset and development of many diseases including metabolic disorders, inflammatory disease, cancer, etc. Changes in protein phosphorylation can be attributed to both changes in phosphorylation events as well as changes due to total protein levels. In order to distinguish the changes in phosphorylation from changes in protein expression, it is important to normalize the signal from phosphorylation over the signal from total protein. For this need, the MILLIPLEX® MAP 2-plex Phospho/Total STAT3 Kit (Cat. No. 48-623MAG) has been developed for the simultaneous detection of phosphorylated STAT3 (Tyr705) and total STAT3 in a single well using the Luminex system.

Specificity

Cross-reactivity between the antibodies and any of the other analytes in this kit is non-detectable or negligible.

application

Intracellular Bead-Based Multiplex Assays using the Luminex technology enables the simultaneous relative quantitation of multiple phosphorylation or total pathway proteins in tissue and cell lysate samples. The analytes available for this multiplex kit are: STAT3 (Total), STAT3 (Tyr705).This is an overnight (4°C) incubation assay.This kit must be run using the provided Assay Buffer.This assay requires 25 μL diluted filtered cell lysate per well.The suggested working range of protein concentration for the assay is 1 to 25 μg of total protein/well (25 μL/well at 40 to 1,000 μg/mL).Analytes available:STAT3 (Total)STAT3 (Tyr705)

Packaging

96-well plate

Legal Information

Luminex is a registered trademark of Luminex Corp
MILLIPLEX is a registered trademark of Merck KGaA, Darmstadt, Germany
xMAP is a registered trademark of Luminex Corp

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

signalword

Danger

Hazard Classifications

Acute Tox. 4 Oral - Aquatic Acute 1 - Aquatic Chronic 2 - Eye Dam. 1 - Skin Irrit. 2

Storage Class

10 - Combustible liquids

Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

Already Own This Product?

Find documentation for the products that you have recently purchased in the Document Library.

Visit the Document Library

Related Content

Cell Signaling Multiplex Assays | MILLIPLEX® Assays

Uncover how cells communicate with MILLIPLEX® cell signaling multiplex assays. Multiplexing with cell signaling phosphoprotein assays based on Luminex® xMAP® technology helps researchers measure phosphoproteins and total proteins within the same or different pathways from a single sample.

Questions

  1. Do you have a protocol for sample preparation to run PBMCs in the Milliplex Cell Signaling assays?

    1 answer

    1. The protocol for sample preparation in the Milliplex Cell Signaling assays has been developed by the R&D team specifically for a custom assay. It is important to note that this protocol has not been tested in all Cell Signaling panels, so volumes and concentrations may need to be optimized for individual studies.

      Here is a short protocol for PBMC cell lysis:
      1. If PBMC cells are frozen, it is recommended to allow cells to recover for 24 hours in complete media as less than 24 hours of recovery can lead to a decrease in signal.
      2. After 24 hours of recovery, count the cells using an appropriate cell counter.
      3. Pellet the PBMC cells at 1000 x g using a tabletop centrifuge for 5 minutes at room temperature.
      4. Remove the supernatant and wash the cells with PBS.
      5. Pellet the PBMC cells again at 1000 x g using a tabletop centrifuge for 5 minutes at room temperature.
      6. Add 10uL of Lysis Buffer (with 2x concentrated protease inhibitors added just prior to use) per 1-2 million cells.
      7. Gently vortex for 30 seconds before transferring the cell lysate into a centrifuge tube.
      8. Gently rock the cell lysate for 10 minutes at 4°C.
      9. Pellet unbroken cells and organelles at 12,000 x g for 10 minutes at 4°C.
      10. Transfer the clear supernatant into a new centrifuge tube.
      11. It is recommended, at least for the first time, to determine the total protein concentration. If not, then it is recommended to run a lysate titration starting at 10uL sample + 15uL of Assay Buffer 1 and performing a 1:1 serial dilution in Assay Buffer 1.

      For the Custom assay, the optimal total protein concentration was found to be 2 to 6 mg/mL. Additionally, it was determined that 10uL of Lysis Buffer per 1 million PBMC cells yields approximately 2 mg/mL, and adding 10uL of this 2mg/mL sample plus 15uL of Assay Buffer yielded good results. It is advised not to dilute samples in Lysis Buffer; rather, dilute in Assay Buffer 1, which lacks strong detergents. If needed, the amount of Lysis Buffer per PBMC cells can be adjusted, for example, by increasing it to 10uL of Lysis Buffer per 2 million PBMC cells.

      Helpful?

Reviews

No rating value

Active Filters

Our team of scientists has experience in all areas of research including Life Science, Material Science, Chemical Synthesis, Chromatography, Analytical and many others.

Contact Technical Service