Kit Summary

To verify simultaneously detect the relative levels of 105 different soluble receptors expressed and released by hematopoietic cells in a single sample. No specialized equipment is necessary.

General Assay Principle

Carefully selected capture antibodies have been spotted in duplicate on nitrocellulose membranes. Cell culture supernatant, cell lysate, or serum samples are diluted and mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture is then incubated with the array. Any cytokine/detection antibody complex present is bound by its cognate immobilized capture antibody on the membrane. Streptavidin-Horseradish Peroxidase and chemiluminescent detection reagents are added, and a signal is produced in proportion to the amount of cytokine bound. Chemiluminescence is detected in the same manner as a Western blot.

 

Kit Contents
  • 2 Hematopoeitic Array and Membranes
  • 2 Common Analyte Array Membranes
  • 4-Well Multi-dish
  • Array Buffers
  • Wash Buffer
  • Detection Antibody Cocktail
  • Streptavidin-HRP
  • Chemiluminescent Detection Reagents
  • Transparency Overlay Template
  • Detailed Protocol

For a complete list of the kit contents and necessary materials, please see the Materials Provided and Other Materials Required sections of the product datasheet.

 
Simultaneously detect the levels of these soluble receptors in a single sample.
Hematopoeitic Array     Common Analytes Array
CD5 Integrin alpha 5/CD49e     ACE HB-EGF
CD6 Integrin alpha 6/CD49f     ADAM8 ICAM-2/CD102
CD30 Integrin alpha 9     ADAM9 IL-1 RII
CD40 Integrin alpha E/CD103     ADAM10 IL-15 R alpha
CD43 Integrin alpha L/CD11a     ALCAM/CD166 Integrin beta 1/CD29
CD48/SLAMF2 Integrin alpha M/CD11b     Amphiregulin Integrin beta 2/CD18
CD59 Integrin alpha X/CD11c     APP (pan) Integrin beta 3/CD61
CD84/SLAMF5 LAG-3     BACE-1 Integrin beta 4/CD104
CD97 MMP-9 (total)     BCAM/CD239 Integrin beta 5
CD163 MMR     C1q R1/CD93 Integrin beta 6
CD229/SLAMF3 Myeloperoxidase     CD9 JAM-A/CD321
Chitinase 3-like 1/YKL40 Resistin     CD23/Fc epsilon RII Lipocalin-2/NGAL
CRP L-Selectin     CD31/PECAM-1 LOX-1/SR-E1/CLEC8A
CRTAM Siglec-5     CD36/SR-B3 MD-1/LY86
CXCL16 Siglec-6     CD40 Ligand MMP-2 (total)
DNAM-1 Siglec-7     CD44H NCAM-1/CD56
DPPIV/CD26 Siglec-9     CD58/LFA-3 NCAM-L1/CD171
IFN-gamma R2 Siglec-10     CD90/Thy1 Osteopontin
IL-2 R alpha TIM-3/KIM-3     CD99 PAR1
IL-2 R beta TLR2/CD282     CD155/PVR Pref-1/DLK-1/FA1
IL-6 R TLR4/CD284     CEACAM-1/CD66a RECK
Integrin alpha 3/CD49c TNF RI     CX3CL1/Fractalkine Stabilin-1
Integrin alpha 4/CD49d TRACP/PAP/ACP5     CXCL8/IL-8 TACE/ADAM17
  TRANCE/RANKL     EMMPRIN/CD147 Thrombospondin-1
  TREM-1     Endoglin/CD105 TIMP-1
        Epiregulin TIMP-2
        Galectin-1 TIMP-3
        Galectin-3 TNF RII
        Galectin-3BP/MAC-2BP  
Preparation and Storage
  • Stability & 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, relative expression levels of human soluble receptors in samples can be determined using the following procedure:

  • Prepare membrane and incubate with prepared sample
  • Incubate the membrane with Detection Antibody Cocktail
  • Incubate the membrane array with Streptavidin-HRP
  • Develop the membrane array with Chemi Reagents 1 and 2
  • Expose the membrane array to autoradiography film
 

 

Reagents Provided
  • 2 Hematopoietic Array Membranes
  • 2 Common Analyte Array Membranes
  • Lysis Buffer 17
  • Array Buffer 1
  • Array Buffer 8
  • Detection Antibody Cocktail H
  • Detection Antibody Cocktail C
  • Streptavidin-HRP
  • Transparency Overlay Template
  • Wash Buffer Concentrate (25X)

 

Other Supplies Required

Reagents

Materials

  • Pipettes and pipette tips
  • Gloves
  • Plastic container 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
  • Absorbent lab wipes (KimWipes® or equivalent)
  • Paper towels
  • X-ray film (Kodak BioMax Light-1) or equivalent
  • Flat-tipped tweezers

Equipment

  • Rocking platform shaker
  • Microcentrifuge
  • Autoradiography cassette
  • Film developer
  • Flatbed scanner with transparency adapter capable of transmission mode
  • Computer capable of running image analysis software and Microsoft Excel

Other Supplies Required for Cell Lysate Samples

  • Phosphate-Buffered Saline (PBS)
  • Lysis buffer (1% Igepal CA-630, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 10 µg/mL Aprotinin, 10 µg/mL Leupeptin, and 10 µg/mL Pepstatin)

Other Supplies Required for Tissue Lysate Samples

  • PBS with protease inhibitors (10 µg/mL Aprotinin, 10 µg/mL Leupeptin, and 10 µg/mL Pepstatin)
  • Triton X-100

 

Procedure Overview

Add 2 mL of Array Buffer 6 to each well of the supplied 4-Well Multi-dish.

Add 2 mL of Array Buffer 6 to each well of the supplied 4-Well Multi-dish

Place each array membrane in a separate well of the 4-Well Multi-dish.

Incubate for one hour on a rocking platform shaker.

Place each array membrane in a separate well of the 4-Well Multi-dish

Add 0.5 mL Array Buffer 4 to each sample.

Adjust volume of each sample to final volume of 1.5 mL with Array Buffer 6.

Visualize using a fluorescence microscope.
Citations:

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

1 Citations: Showing 1 - 1

  1. Signaling via PI3K/FOXO1A pathway modulates formation and survival of human embryonic stem cell-derived endothelial cells.
    Authors: Merkely B, Gara E, Lendvai Z, Skopal J, Leja T, Zhou W, Kosztin A, Varady G, Mioulane M, Bagyura Z, Nemeth T, Harding S, Foldes G
    Stem Cells Dev, 2015;24(7):869-78. 2015
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