Caspase 9 (Phospho-Thr125) ELISA Kit

Name: Caspase 9 (Phospho-Thr125) ELISA Kit
SKU: DEIA-XYA885
Rating: 5 (1 reviews)

Regulatory status: For research use only, not for use in diagnostic procedures.

COA

SDS

Datasheet

Summary

Sample

cultured cells

Species Reactivity

Human; Mouse; Rat

Detection Method

iELISA

Intended Use

The Caspase 9 (Phospho-Thr125) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor Caspase 9 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Caspase 9 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Caspase 9 phosphorylation.

Contents of Kit

1. 96-Well Cell Culture Clear-Bottom Microplate, 1 Plate
2. 10× TBS, 24 ml (10×)
3. Quenching Buffer, 24 ml (1×)
4. Blocking Buffer, 50 ml (1×)
5. 15× Wash Buffer, 50 ml (15×)
6. 100× Anti-Phospho-Caspase 9 (Thr125) Antibody, 60 μl (100×)
7. 100× Anti-Caspase 9 Antibody, 60 μl (100×)
9. 100× Anti-GAPDH Antibody (Mouse Monoclonal), 60 μl (100×)
10. HRP-Conjugated Anti-Rabbit IgG Antibody, 12 ml (1×)
11. HRP-Conjugated Anti-Mouse IgG Antibody, 12 ml (1×)
12. Primary Antibody Diluent, 12 ml (1×)
13. Ready-to-Use Substrate, 12 ml (1×)
14. Stop Solution, 12 ml (1×)
15. Crystal Violet Solution, 12 ml (1×)
16. SDS Solution, 24 ml (1×)
17. Adhesive Plate Seals, 2 Seals

Storage

Upon receipt, the kit should be stored at 4°C. The un-opened kit will be stable for up to 6 months from the date of shipment if stored at 4°C. Diluted Anti-Phospho-Caspase 9 (Thr125) Antibody, Caspase 9 Antibody and diluted Anti- GAPDH Antibody can each be stored at 4°C for up to two weeks. HRP-Conjugated Anti-Rabbit IgG Antibody and HRP-Conjugated Anti-Mouse IgG Antibody will be stable at 4°C for up to six months. The SDS Solution and Quenching Buffer should be stored at room temperature or warmed up to room temperature if stored at 4°C.

General Description

The Colorimetric Cell-Based ELISA Kit allows for the detection of various target proteins and the effects that certain stimulation conditions have on target protein expression in different cell lines. Qualitative determination of target protein concentration is achieved by an indirect ELISA format. In essence, the target protein is captured by target-specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° antibody can catalyze a colorimetric reaction upon substrate addition.
Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are described: 1) a monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. 2) Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet wholecell staining method is used to determine cell density. After staining, the results can be analyzed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. 3) If a phosphorylated target is being detected, an antibody against the nonphosphorylated counterpart will be provided for normalization purposes. The absorbance values obtained for the non-phosphorylated target can be used to normalize the absorbance values for the phosphorylated target.

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Documents

pdf COA

pdf SDS

pdf Datasheet

QA & Reviews

Customer Reviews

References

Gene expression signature of primary imatinib-resistant chronic myeloid leukemia patients

LEUKEMIA

Authors: Frank, O.; Brors, B.; Fabarius, A.; Li, L.; Haak, M.; Merk, S.; Schwindel, U.; Zheng, C.

Abstract

Although the selective tyrosine kinase inhibitor imatinib is successfully used in the treatment of chronic myeloid leukemia (CML), inherent mechanisms confer primary resistance to leukemic patients. In order to search for potentially useful genes in predicting cytogenetic response, a retrospective gene expression study was performed. Leukocyte RNA isolated before imatinib from interferon-alpha-pretreated chronic phase CML patients (n = 34) with or without major cytogenetic remission (<= 35% Philadelphia (Ph)(+) metaphases) during the first year of treatment was comparatively analyzed using Affymetrix U133A chips. Using support vector machines for gene classification, an outcome-specific gene expression signature consisting of 128 genes was identified. Comparative expression data of specific genes point to changes in apoptosis (e.g. casp9, tumor necrosis factor receptor-associated protein 1, hras), DNA repair (msh3, ddb2), oxidative stress protection (glutathione synthetase, paraoxonase 2, vanin 1) and centrosomes (inhibitor of differentiation-1) within primary resistant patients. Independent statistical approaches and quantitative real-time reverse transcriptase-polymerase chain reaction studies support the clinical relevance of gene profiling. In conclusion, this study establishes a candidate predictor of imatinib resistance in interferon-alpha-pretreated CML patients to be subjected to future investigation in a larger independent patient cohort. The resulting expression signature point to involvement of BCR-ABL-independent mechanisms of resistance.

Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine

SCIENTIFIC REPORTS

Authors: Kopp, Sascha; Slumstrup, Lasse; Corydon, Thomas J.; Sahana, Jayashree; Aleshcheva, Ganna; Islam, Tawhidul; Magnusson, Nils E.; Wehland, Markus; Bauer, Johann; Infanger, Manfred; Grimm, Daniela

Abstract

Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h-and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA, and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD, and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking.