Do I need to use the protease inhibitors (e.g. aprotinin, Ipegal) if I only plan to use cell supernatants?

Name: Proteome Profiler Human Pluripotent Stem Cell Array Kit
Brand: R&D Systems
SKU: ARY010

The proteinase inhibitors are recommended for cell and tissue lysates only. You do not need them for cell culture supernates.

Proteome Profiler Human Pluripotent Stem Cell Array Kit

R&D Systems, Inc. a Bio-Techne Brand

Catalog # ARY010

Citations (24)

Product Datasheet / COA / SDS

Contains 8 membranes - each spotted in duplicate with 15 different stem cell marker antibodies

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Setting the standard in quality research reagents

Summary for Proteome Profiler Human Pluripotent Stem Cell Array Kit

Kit Summary

A membrane-based antibody array for the simultaneous detection of 15 established stem cell markers.

Troubleshooting Guide

Key Benefits

Verifies stem cell identity
Detects 15 stem cell markers simultaneously
Easier to perform than a Western blot
Requires no specialized equipment
The array protocol can now be performed with a smaller sample size! Save more of your valuable stem cell cultures by simultaneously detecting pluripotency and germ layer markers using lysate from only 1 well of a 24-well plate.

Why Use an Antibody Array to Detect Multiple Stem Cell Markers in Parallel?

Examining the expression profile of stem cells can be expensive, time-consuming, and can require specialized equipment. For example, microarrays are expensive and require specialized equipment. Similar expression analyses can be achieved by performing multiple immunoprecipitaions and Western blots, although these techniques are also time-consuming.

To address this need, R&D Systems offers the Proteome Profiler Human Pluripotent Stem Cell Antibody Array Kit. This rapid, sensitive, and economical multiplex assay allows users to determine if their cells express markers of pluripotency consistent with undifferentiated cells or if the cells express markers consistent with differentiation towards lineages such as the trophectoderm, ectoderm, mesoderm, and endoderm. No specialized equipment is needed as data are obtained by chemiluminescence.

The Proteome Profiler Human Pluripotent Stem Cell Antibody Array Kit:

Is a sensitive multiplex tool to verify stem cell identity using 15 stem cell markers.
Has a protocol that can be completed with just 5.5 hours of hands-on time.
Is more efficient than other techniques used to establish pluripotency.
Does not require specialized equipment.

Kit Contents

Each kit contains eight nitrocellulose membranes spotted with 15 different antibodies printed in duplicate to detect stem cell markers. Buffers, biotinylated detection antibodies, streptavidin-HRP, and chemiluminescent reagents are included to enable the detection of stem cell markers from cell lysates.

8-well Rectangular Multi-dish
Human Pluripotent Stem Cell Array - 8 nitrocellulose membranes each spotted with 15 different antibodies to stem cell markers printed in duplicate
Array Buffer 1
Array Buffer 2 Concentrate (5X)
Array Buffer 3
Chemi Reagent 1
Chemi Reagent 2
Detection Antibody Cocktail, Human Pluripotent Stem Cell Array
Lysis Buffer 16
Streptavidin-HRP
Transparency Overlay Template
Wash Buffer Concentrate (25X)

Analytes Detected by this kit:

Oct-3/4
Nanog
SOX2
E-Cadherin
Alpha-Fetoprotein (AFP)
GATA-4
HNF-3 beta/FoxA2
PDX-1/IPF1
SOX17
Otx2
TP63/TP73L
Goosecoid (Gsc)
Snail
VEGF R2/KDR/Flk-1
HCG

Stability and Storage

Store the unopened kit at 2 ^°C to 8 ^°C. Do not use past kit expiration date.

Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of pre-implantation embryos. Induced pluripotent stem (iPS) cells can be generated by somatic cell reprogramming following the exogenous expression of specific transcription factors (Oct-3/4, KLF4, SOX2, and c-Myc). These cell types are capable of unlimited, undifferentiated proliferation in vitro and still maintain the capacity to differentiate into a wide variety of somatic cells. In this capacity, pluripotent stem cells have widespread clinical potential for the treatments of heart disease, diabetes, spinal cord injury, and a variety of neurodegenerative disorders.

R&D Systems offers a wide range of products to support pluripotent stem cell culture and differentiation. Mouse embryonic fibroblasts may be used to maintain and expand pluripotent stem cells in an undifferentiated state. We also offer defined culture media, which are specifically optimized for use with human or rodent pluripotent stem cells. In addition, R&D Systems offers a variety of products to assess differentiation status and identify specific stem cell types of interest, including panels of marker antibodies, primer pairs, multi-color flow cytometry kits, and specialized verification kits.

Product Specifications for Proteome Profiler Human Pluripotent Stem Cell Array Kit

Species

Human

Scientific Data Examples for Proteome Profiler Human Pluripotent Stem Cell Array Kit

	siRNA-mediated Knockdown of PDGF R alpha, PDGF R beta, and cAbl Upregulates Expression of Pluripotent, Mesoderm, and Endoderm Markers. Small interfering RNAs (siRNAs) were transfected into mesenchymal stem cells by electroporation. Knockdown was verified by RT-PCR and Western blot analysis (data not shown here). Cell lysates from siRNA-treated cells were used in the Proteome Profiler Human Pluripotent Stem Cell Antibody Array (Catalog # ARY010) to examine relative expression levels of mesoderm, endoderm, and pluripotent markers. The images represent nitrocellulose membranes following chemiluminescent detection of bound analytes. Each of the array markers has duplicate spots, which are boxed to highlight their identity. A. Scrambled (Scr) siRNA-treated mesenchymal stem cells. B. Knockdown of PDGF R alpha upregulated pluripotent markers (Oct-3/4, Nanog, SOX2, and E-Cadherin) as well as markers of the mesoderm and endoderm compared to Scr. C. Knockdown of PDGF R beta upregulated expression of Snail, SOX17, VEGF R2, Oct-3/4, and Nanog compared to both Scr and PDGF R alpha knockdowns. D. Knockdown of cAbl upregulated pluripotent markers (Nanog, Oct-3/4, E-Cadherin, and SOX2) compared to Scr in addition to markers of the endoderm and mesoderm.Adapted from Ball, S.G. et al. (2012) Stem Cells 30:548.
	Small Sample Lysate Volume Used to Detect Germ Layer Markers in Differentiated Human Pluripotent Stem Cells. BG01V human embryonic stem cells were differentiated into mesoderm, endoderm, and ectoderm using the differentiation supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B). Following differentiation in a 24-well plate, a single well of confluent cells was lysed in 100 µL of lysis buffer and analyzed using the Proteome Profiler Human Pluripotent Stem Cell Antibody Array (Catalog # ARY010). The differentiation supplements upregulated expression of the germ layer markers (A) Snail, (B) Sox17, and (C) Otx2 compared to undifferentiated cells. Immunocytochemistry for the same markers (Snail, Catalog # AF3639; Sox17, Catalog # AF1924; and Otx2, Catalog # AF1979; red) followed by staining with the appropriate NorthernLights™-conjugated Secondary Antibodies and counterstaining with DAPI (blue) is consistent with data obtained by array analysis.

Preparation & Storage

Shipping Conditions	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Storage	Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, the expression of stem cell markers is assessed using the following procedure:

Incubate cell lysates with the antibody-spotted array
Add biotinylated detection antibodies
Add streptavidin-HRP reagents
Detect signal by chemiluminescence

View Graphic Procedure

Reagents Provided

Reagents Supplied in the Proteome Profiler Human Pluripotent Stem Cell Antibody Array Kit (Catalog # ARY010)

8-well Rectangular Multi-dish
Human Pluripotent Stem Cell Array - 8 nitrocellulose membranes each containing 15 different antibodies to stem cell markers printed in duplicate
Array Buffer 1
Array Buffer 2 Concentrate (5X)
Array Buffer 3
Chemi Reagent 1
Chemi Reagent 2
Detection Antibody Cocktail, Human Pluripotent Stem Cell Array
Lysis Buffer 16
Streptavidin-HRP
Transparency Overlay Template
Wash Buffer Concentrate (25X)

Other Supplies Required

Reagents

Aprotinin
Leupeptin (Catalog # 1167)
Pepstatin (Catalog # 1190)
Deionized or distilled water

Materials

Pipettes and pipette tips
Gloves
Plastic containers with the capacity to hold 50 mL (for washing the arrays)
Plastic transparent sheet protector (trimmed to 10 cm x 12 cm and open on three sides)
Plastic wrap
Paper towels
Absorbent lab wipes
Autoradiography cassette
X-ray film (Kodak^® BioMax^™ Light-1) or equivalent
Flat-tipped tweezers

Equipment

Rocking platform shaker
Microcentrifuge
Film developer
Flatbed scanner with transparency adapter capable of transmission mode

Procedure Overview

Add 1 mL of Array Buffer to each well of an 8-well multi-dish.

Place each membrane that will be used in one well of the 8-well multi-dish. The number on the membrane should be facing upward.

Incubate for one hour on a rocking platform shaker. Orient the tray so that each membrane rocks end to end in its well.

Replace the Array Buffer with prepared samples containing cell extract, Array Buffer 1, and Lysis Buffer for a final volume of 1 mL.

Incubate overnight at 2 °C to 8 °C on a rocking platform.

Wash each array 3 times with Wash Buffer in a separate container.

Wash each well of the 8-well multi-dish.

Replace the Array Buffer with prepared samples containing cell extract, Array Buffer 1, and Lysis Buffer for a final volume of 1 mL

Add 1 mL of diluted Biotinylated Detection Antibody Cocktail into each well.

Add the array to the well.

Incubate for 1 hour on a rocking platform.

Wash each array 3 times with Wash Buffer in a separate container.

Wash each well of the 8-well multi-dish.

Add 1 mL of diluted Biotinylated Detection Antibody Cocktail into each well

Add 1 mL of diluted Streptavidin-HRP to each well.

Add the array to the diluted Streptavidin-HRP.

Incubate for 30 minutes on a rocking platform.

Wash each membrane 3 times with Wash Buffer in a separate container.

Wash each well of the 8-well multi-dish.

Add 1 mL of diluted Streptavidin-HRP to each well

Place the array on a plastic sheet protector.

Add 0.5 mL of the prepared Chemi Reagent Mix evenly onto the array.

Cover the array with the top sheet of the plastic protector and incubate for 1 minute.

Blot off excess Chemi Reagent Mix.

Wrap the array and sheet protector in plastic wrap.

Place the wrapped array in an autoradiography film cassette and expose to film.

Place the array on a plastic sheet protector

Citations for Proteome Profiler Human Pluripotent Stem Cell Array Kit (24)

Showing 1 - 10 of 24 citations Showing All

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Citations are publications that use Bio-Techne products. Selected citations for Proteome Profiler Human Pluripotent Stem Cell Array Kit include:

Species: Human
Sample Types: Cell Lysates

L László et al. (2022), A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System International Journal of Molecular Sciences, 23(15).
PMID: 35955935
Species: Human
Sample Types: Cell Lysates

N Kfoury-Bea et al. (2022), The H3K27M mutation alters stem cell growth, epigenetic regulation, and differentiation potential Bmc Biology, 20(1):124.
PMID: 35637482
Species: Human
Sample Types: Cell Lysates

V Pecce et al. (2021), The role of FOSL1 in stem-like cell reprogramming processes Scientific Reports, 11(1):14677.
PMID: 34282187
Species: Human
Sample Types: Cell Lysates

AE Herzog et al. (2021), The IL-6R and Bmi-1 axis controls self-renewal and chemoresistance of head and neck cancer stem cells Cell Death Disease, 2021;12(11):988.
PMID: 34689150
Species: Human
Sample Types: Whole Cells

J Strnadel et al. (2020), Generation of ORIONi001-A induced pluripotent stem cell line for in vitro modeling of sporadic form of amyotrophic lateral sclerosis Stem Cell Res, 48(0):101981.
PMID: 32919353
Species: Human
Sample Types: Cell Lysates

H Solís-Chag et al. (2019), Purinergic Signaling Pathway in Human Olfactory Neuronal Precursor Cells Stem Cells Int, 2019(0):2728786.
PMID: 31065271
Species: Human
Sample Types: Cell Lysates

MR Guda et al. (2019), GLUT1 and TUBB4 in Glioblastoma Could be Efficacious Targets Cancers (Basel), 11(9).
PMID: 31491891
Species: Human
Sample Types: Cell Lysates

A Gambacurta et al. (2019), Human osteogenic differentiation in Space: proteomic and epigenetic clues to better understand osteoporosis Sci Rep, 9(1):8343.
PMID: 31171801
Species: Human
Sample Types: Cell Lysates

D Ravindran et al. (2018), CDK1 interacts with Sox2 and promotes tumor initiation in human melanoma Cancer Res., 0(0).
PMID: 30297536
Species: Human
Sample Types: Cell Lysates

MR Guda et al. (2018), Targeting PDK4 inhibits breast cancer metabolism Am J Cancer Res, 8(9):1725-1738.
PMID: 30323966
Species: Human
Sample Types: Cell Culture Supernates

(2016), Tumor-Activated Mesenchymal Stromal Cells Promote Osteosarcoma Stemness and Migratory Potential via IL-6 Secretion PLoS ONE, 11(11):e0166500.
PMID: 27851822
Species: Human
Sample Types: Cell Lysates

Swapna Asuthkar (2016), Hand1 overexpression inhibits medulloblastoma metastasis Biochem Biophys Res Commun, 0(0).
PMID: 27297109
Species: Human
Sample Types: Cell Lysates

G Hamilton et al. (2016), Small cell lung cancer: Circulating tumor cells of extended stage patients express a mesenchymal-epithelial transition phenotype Cell Adh Migr, 10(4):360-7.
PMID: 26919626
Species: Human
Sample Types: Cell Lysates

James M et al. (2015), Characterization and propagation of tumor initiating cells derived from colorectal liver metastases: trials, tribulations and a cautionary note. PLoS ONE, 10(2):e0117776.
PMID: 25658706
Species: Human
Sample Types: Cell Lysates

Bobis-Wozowicz S et al. (2015), Human Induced Pluripotent Stem Cell-Derived Microvesicles Transmit RNAs and Proteins to Recipient Mature Heart Cells Modulating Cell Fate and Behavior. Stem Cells, 33(9):2748-61.
PMID: 26031404
Valverde A et al. (2015), Simultaneous inhibition of EGFR/VEGFR and cyclooxygenase-2 targets stemness-related pathways in colorectal cancer cells. PLoS ONE, 10(6):e0131363.
PMID: 26107817
Species: Human
Sample Types: Cell Lysates

Canham et al. (2015), Increased cycling cell numbers and stem cell associated proteins as potential biomarkers for high grade human papillomavirus+ve pre-neoplastic cervical disease. PLoS ONE, 9(12):e115379.
PMID: 25531390
Species: Human
Sample Types: Cell Lysates

Labsch S et al. (2014), Sulforaphane and TRAIL induce a synergistic elimination of advanced prostate cancer stem-like cells. Int J Oncol, 44(5):1470-80.
PMID: 24626333
Species: Human
Sample Types: Whole Cells

Liu L et al. (2014), Triptolide reverses hypoxia-induced epithelial-mesenchymal transition and stem-like features in pancreatic cancer by NF-kappaB downregulation. Int J Cancer, 134(10):2489-503.
PMID: 24615157
Species: Human
Sample Types: Cell Lysates

Hage C et al. (2013), The novel c-Met inhibitor cabozantinib overcomes gemcitabine resistance and stem cell signaling in pancreatic cancer. Cell Death Dis, 4(0):e627.
PMID: 23661005
Species: Human
Sample Types: Cell Lysates

Raynaud C et al. (2013), Human embryonic stem cell derived mesenchymal progenitors express cardiac markers but do not form contractile cardiomyocytes. PLoS ONE, 8(1):e54524.
PMID: 23342164
Species: Human
Sample Types: Cell Lysates

Leis O et al. (2012), Sox2 expression in breast tumours and activation in breast cancer stem cells. Oncogene, 31(11):1354-65.
PMID: 21822303
Species: Human
Sample Types: Cell Lysates

Ball SG et al. (2012), Inhibition of platelet-derived growth factor receptor signaling regulates Oct4 and Nanog expression, cell shape, and mesenchymal stem cell potency. Stem Cells, 30(3):548-60.
PMID: 22213560
Species: Human
Sample Types: Cell Lysates

Wortmann A et al. (2011), Cellular settings mediating Src Substrate switching between focal adhesion kinase tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) tyrosine 734. J. Biol. Chem., 286(49):42303-15.
PMID: 21994943

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Showing 1 - 10 of 24 citations Showing All

FAQs for Proteome Profiler Human Pluripotent Stem Cell Array Kit

Do I need to use the protease inhibitors (e.g. aprotinin, Ipegal) if I only plan to use cell supernatants?

The proteinase inhibitors are recommended for cell and tissue lysates only. You do not need them for cell culture supernates.

Proteome Profiler Human Pluripotent Stem Cell Array Kit

R&D Systems, Inc. a Bio-Techne Brand

Key Product Details

Summary for Proteome Profiler Human Pluripotent Stem Cell Array Kit

Key Benefits

Why Use an Antibody Array to Detect Multiple Stem Cell Markers in Parallel?

The Proteome Profiler Human Pluripotent Stem Cell Antibody Array Kit:

Stability and Storage

Product Specifications for Proteome Profiler Human Pluripotent Stem Cell Array Kit

Scientific Data Examples for Proteome Profiler Human Pluripotent Stem Cell Array Kit

Preparation & Storage

Assay Procedure

Reagents Supplied in the Proteome Profiler Human Pluripotent Stem Cell Antibody Array Kit (Catalog # ARY010)

Reagents

Materials

Equipment

Citations for Proteome Profiler Human Pluripotent Stem Cell Array Kit (24)

FAQs for Proteome Profiler Human Pluripotent Stem Cell Array Kit

Product Documents for Proteome Profiler Human Pluripotent Stem Cell Array Kit

Product Datasheets

COA

Certificate of Analysis

SDS

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