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Assay Format

Pathway:

Akt Signaling
HER/ErbB Signaling
IGF Signaling
JAK/STAT Signaling
MAPK Signaling
NFkB Signaling
PI3K-AKT Signaling
Wnt/beta-Catenin Signaling

Number of Targets Detected:

Species Detected:

Human
Mouse

Compatible Sample Types:

Cell Culture Supernatants
Cell Lysates
Plasma
Serum
Tissue Lysates

Design Principle:

Sandwich-based

Method of Detection:

Chemiluminescence

Quantitative/Semi-Quantitative:

Semi-Quantitative

Solid Support:

Membrane

Product Specifications

Size:

2, 4, or 8 Sample Kit

Protein Information

Gene Symbols:

Product Features

Easy to use
No specialized equipment needed
Compatible with nearly any liquid sample
Proven technology (many publications)
Highly sensitive (pg/ml)
Sandwich ELISA specificity
Higher density than ELISA, Western blot or bead-based multiplex

Target Names

Scroll over each target protein for more information
Akt (P-Ser473) (PKB (P-Ser473)) Gene Symbol: AKT1 PKB RAC Gene ID: 207 Accession #: P31749 ELISA Kit: RayBio Human/Mouse/Rat Phospho-Akt (S473) ELISA	CREB (P-Ser133) Gene Symbol: CREB1 Gene ID: 1385 Accession #: P16220 ELISA Kit: RayBio Human Phospho-CREB (S133) ELISA	ERK1 (P-T202/Y204)/ERK2 (P-Y185/Y187) Gene Symbol: MAPK1 ERK2 PRKM1 PRKM2 Gene ID: 5594 Accession #: P28482 ELISA Kit: RayBio Human/Mouse/Rat Phospho-Erk1 (T202/Y204) + Phospho-Erk2 (Y185/Y187) and Total Erk1/2 ELISA	GSK3a (P-Ser21) Gene Symbol: GSK3A Gene ID: 2931 Accession #: P49840 ELISA Kit: RayBio Human/Mouse/Rat Phospho-GSK3a (S21) ELISA	GSK3b (P-Ser9) Gene Symbol: GSK3B Gene ID: 2932 Accession #: P49841 ELISA Kit: RayBio Human Phospho-GSK3b (S9) ELISA
HSP27 (P-Ser82) Gene Symbol: HSPB1 HSP27 HSP28 Gene ID: 3315 Accession #: P04792 ELISA Kit: RayBio Human Phopspho-HSP27 (S82) ELISA	JNK (P-Thr183) Gene Symbol: MAPK8 JNK1 PRKM8 SAPK1 SAPK1C Gene ID: 5599 Accession #: P45983 ELISA Kit: RayBio Human/Mouse/Rat Phospho-JNK (T183/Y185) ELISA	MEK (P-Ser217/Ser221) Gene Symbol: MAP2K1 MEK1 PRKMK1 Gene ID: 5604 Accession #: Q02750 ELISA Kit: RayBio Human/Mouse/Rat Phospho-MEK1 (S217/S221) ELISA	MKK3 (P-Ser189) Gene Symbol: MAP2K3 MEK3 MKK3 PRKMK3 SKK2 Gene ID: 5606 Accession #: P46734 ELISA Kit: RayBio Human/Mouse/Rat Phospho-MKK3 (S189) ELISA	MKK6 (P-Ser207) Gene Symbol: MAP2K6 MEK6 MKK6 PRKMK6 SKK3 Gene ID: 5608 Accession #: P52564 ELISA Kit: RayBio Human/Mouse/Rat Phospho-MKK6 (S207) ELISA
MSK2 (P-Ser360) Gene Symbol: RPS6KA4 MSK2 Gene ID: 8986 Accession #: O75676 ELISA Kit: RayBio Human Phospho-MSK2 (S360) ELISA	mTOR (P-Ser2448) Gene Symbol: MTOR FRAP FRAP1 FRAP2 RAFT1 RAPT1 Gene ID: 2475 Accession #: P42345 ELISA Kit: RayBio Human Phospho-mTOR (S2448) ELISA	p38 (P-Thr180/Tyr182) Gene Symbol: MAPK14 CSBP CSBP1 CSBP2 CSPB1 MXI2 SAPK2A Gene ID: 1432 Accession #: Q16539 ELISA Kit: RayBio Human/Mouse Phospho-p38 alpha (T180/Y182) ELISA	P53 (P-Ser15) Gene Symbol: TP53 P53 Gene ID: 7157 Accession #: P04637 ELISA Kit: RayBio Human/Mouse/Rat Phospho-P53 (S15) ELISA	P70S6K (P-Thr421/Ser424) Gene Symbol: RPS6KB1 STK14A Gene ID: 6198 Accession #: P23443 ELISA Kit: RayBio Human/Mouse/Rat Phospho-P70S6K (T421/S424) ELISA
RSK1 (P-Ser380) Gene Symbol: RPS6KA1 MAPKAPK1A RSK1 Gene ID: 6195 Accession #: Q15418 ELISA Kit: RayBio Human Phosph-RSK1 (S380) ELISA	RSK2 (P-Ser386) Gene Symbol: RPS6KA3 ISPK1 MAPKAPK1B RSK2 Gene ID: 10432 Accession #: P51812

Application Notes

Suggested Application

Multiplexed Protein Detection; Detection of Relative Protein Expression; Detecting Patterns of Cytokine Expression; Biomarker/ Key Factor Screening; Identifying Key Factors; Confirming a Biological Process

Kit Components

Human/Mouse MAPK Phosphorylation Antibody Array C1 Membranes
Blocking Buffer
Detection Antibody Cocktail
1,000X HRP-Anti-Rabbit-IgG Concentrate
20X Wash Buffer I Concentrate
20X Wash Buffer II Concentrate
2X Cell Lysis Buffer Concentrate
Detection Buffer C
Detection Buffer D
8-Well Incubation Tray w/ Lid
Protease Inhibitor Cocktail
100x Phosphatase Inhibitor Cocktail I
Phosphatase Inhibitor Cocktail II
Plastic Sheets
Array Map Template
Manual

Other Materials Required

Pipettors, pipet tips and other common lab consumables
Orbital shaker or oscillating rocker
Tissue Paper, blotting paper or chromatography paper
Adhesive tape or Saran Wrap
Distilled or de-ionized water
A chemiluminescent blot documentation system (such as UVP??s ChemiDoc-It^® or EpiChem II Benchtop Darkroom or GE's ImageQuant^™ LAS 4000 or Amersham Imagers 600 and 680), X-ray Film and a suitable film processor, or another chemiluminescent detection system.

Protocol Outline

Block membranes
Incubate with Sample
Incubate with Detection Antibody Cocktail
Incubate with HRP-Conjugated anti-IgG
Incubate with Detection Buffers
Image with chemiluminescent imaging system
Perform densitometry and analysis

Typical Data

Figure 1 - HeLa cells were grown to 80% confluency and then serum starved overnight. Cells were either untreated (bottom panel) or treated (top panel) with 250 nM PMA for 20 minutes. Data shown are from a 20 second exposure using a chemiluminescence imaging system.

Figure 2 - HepG2 cells were grown to 80% confluency and then serum starved overnight. Cells were either untreated (bottom panel) or treated (top panel) with 25 ng/mL of recombinant human IL-1β for 30 minutes. Data shown are from a 20 second exposure using a chemiluminescence imaging system.

Storage/Stability

For best results, store the entire kit frozen at -20^°C upon arrival. Stored frozen, the kit will be stable for at least 6 months which is the duration of the product warranty period. Once thawed, store array membranes and 1X Blocking Buffer at -20^°C and all other reagents undiluted at 4^°C for no more than 3 months.

Poling, Holly M., et al. "Mechanically induced development and maturation of human intestinal organoids in vivo." Nature biomedical engineering 2.6 (2018): 429-442.
Species: Human
Sample type: Tissue Lysate
Xie Z, Peng M, Lu R, Meng X, Liang W, Li Z, Qiu M, Zhang B, Nie G, Xie N, Zhang H, Prasad PN. Black phosphorus-based photothermal therapy with aCD47-mediated immune checkpoint blockade for enhanced cancer immunotherapy. Light Sci Appl. 2020 Sep 15;9:161. doi: 10.1038/s41377-020-00388-3. PMID: 33014356; PMCID: PMC7492464.
Species: Mouse
Sample type: Tissue Lysate
Yu S., Zhong Q., Chen R., et al. CRLF1 promotes malignant phenotypes of papillary thyroid carcinoma by activating the MAPK/ERK and PI3K/AKT pathways. Cell Death Dis. 2018 Mar 7;9(3):371. doi: 10.1038/s41419-018-0352-0.
Species: Human
Sample type: Cell Lysate (Human thyroid cell lines, normal and cancerous; IHH-4, PTC, etc)
Yu, Shi-Tong, et al. "CRLF1 promotes malignant phenotypes of papillary thyroid carcinoma by activating the MAPK/ERK and PI3K/AKT pathways." Cell death & disease 9.3 (2018): 1-13.
Species: Human
Sample type: Cell Lysate
Schuett J., Schuett H., Oberoi R., et al. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells. FASEB J. 2017 Mar 8. pii: fj.201600729R. doi: 10.1096/fj.201600729R
Species: Human
Sample type: Cell Lysate (Human umbilical cord endothelial cells)
Schuett, Jutta, et al. "NADPH oxidase NOX2 mediates TLR2/6?dependent release of GM?CSF from endothelial cells." The FASEB Journal 31.6 (2017): 2612-2624.
Species: Human
Sample type: Cell Lysate
Yang Z, Zou W, Pan Y, Yong KT, Li L, Wang X, Liu D, Chen T, Xue D, Lin G. PEGylated CuInS2/ZnS quantum dots inhibit neurite outgrowth by downregulating the NGF/p75NTR/MAPK pathway. Ecotoxicol Environ Saf. 2021 Jan 1;207:111378. doi: 10.1016/j.ecoenv.2020.111378. Epub 2020 Oct 3. PMID: 33022524.
Species: Rat
Sample type: Cell Lysate
Dhani, Shanel, et al. "Fusaric acid alters Akt and ampk signalling in c57bl/6 mice brain tissue." Food and Chemical Toxicology (2020): 111252.
Species: Mouse
Sample type: Tissue Lysate (brain tissue)
Sarsour, Ehab H., et al. "Arachidonate 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid contribute to stromal aging-induced progression of pancreatic cancer." Journal of Biological Chemistry 295.20 (2020): 6946-6957.
Species: Human
Sample type: Cell Lysate (pancreatic cells)
L'Huillier C, Jarbeau M, Achamrah N, et al. Glutamine, but not Branched-Chain Amino Acids, Restores Intestinal Barrier Function during Activity-Based Anorexia. Nutrients. 2019;11(6):1348. Published 2019 Jun 15. doi:10.3390/nu11061348
Species: Mouse
Sample type: Tissue Lysate (Colon tissue )

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We compared the MAPK expression patterns in untreated and doxorubicin-treated mouse hearts. Heart muscle tissue extract were assayed at mid-range protein amounts loaded.

Alexi Kiss
George Washington University 12/29/2022, 01:53 PM

Used for human squamous cell carcinoma cell lines.
Worked great, in this experiment we captured the signals on X-ray films and quantified the results using ImageJ software.

Alexi Kiss
George Washington University 12/29/2022, 01:33 PM

1.Experimental conditions and sample type
a. In vitro: Human breast cancer cell line (hBCC)
b. In vivo: hBCC grew up in nude mouse bone + mouse bone (human/mouse protein mixed)

Pros: pAKT1, pCREB, pGSK3b,p70S6K, pRSK1,2, pMEK1, pp38, and pMKK3,6 are good. The experimental process is very easy. Easy to reproduce the results

Cons: pERK1/2 is a signal relatively weak compared to the Cell signaling antibody.

Yubei-BJH
Yale University 03/17/2021, 10:33 AM

This assay is very straightforward and provides good results for brain samples.

Maria Rodrigues
Emory 12/08/2020, 11:57 AM

These arrays are what I was looking for. They are easy to use, very clean membranes with super sharp results. These arrays are a great second step for large scale analysis such RNAseq.

Ehab Sarsour
The University of Iowa 05/06/2019, 01:38 PM

Easy to use and convenient way to look at multiple targets.

Harsh Sharthiya
Midwestern University 03/26/2019, 07:00 PM

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