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Proteome Profiler Human Ubiquitin Array

Contains 4 membranes-each spotted in duplicate with 49 different antibodies to ubiquitin target proteins
Proteome Profiler Human Ubiquitin Array
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ARY027
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Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, relative expression levels of ubiquitination of human proteins 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
 
Kit Contents
  • Rectangular 4-Well Multi-dish
  • 4 Human Ubiquitin Array nitrocellulose membranes spotted with 49 different antibodies to human ubiquitin target proteins
  • Lysis Buffer 6
  • Array Buffer 1
  • Array Buffer 2
  • Chemi Reagent 1
  • Chemi Reagent 2
  • Detection Antibody Human Ubiquitin Array
  • Streptavidin-HRP
  • Transparency Overlay Template
  • Wash Buffer Concentrate (25X)

 

Other Supplies Required

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)
  • Aprotinin
  • Leupeptin (Catalog # EI002)
  • Pepstatin (Catalog # EI003)

 

Procedure Overview

R&D Systems Protocol for Multiple Analyte Detection Using the Proteome Profiler Human XL Oncology Array Kit, Panel A (Catalog # ARY026)

Add 2 mL of Array Buffer 1 to each well of the supplied 4-Well Multi-dish.

ARY027 Step 1

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

Incubate for one hour on a rocking platform shaker.

ARY027 Step 2

Adjust volume of each sample to final volume of 1.5 mL with Array Buffer 1.

ARY027 Step 3

Replace the Array Buffer 1 in each well of the 4-Well Multi-dish with prepared samples.

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

Wash each array membrane 3 times with 1X Wash Buffer in a separate container.

Wash each well of the 4-Well Multi-dish with 1X Wash Buffer.

ARY027 Step 4

Add 15 µL of Detection Antibody to 1.5 mL of Array Buffer 2 for each array.

ARY027 Step 5

Pipette 1.5 mL diluted Detection Antibody Cocktail into each well of the 4-Well Multi-dish.

ARY027 Step 6

Return each array membrane to the 4-Well Multi-dish containing diluted Detection Antibody Cocktail.

Incubate for one hour on a rocking platform shaker.

Wash each array membrane 3 times with 1X Wash Buffer in a separate container.

Wash each well of the 4-Well Multi-dish with 1X Wash Buffer.

ARY027 Step 7

Add 2 mL of diluted Streptavidin-HRP to each well of the 4-Well Multi-dish.

Place the array membrane in the diluted Streptavidin-HRP solution.

Incubate for 30 minutes on a rocking platform shaker.

Wash each array membrane 3 times with 1X Wash Buffer in a separate container.

ARY027 Step 8

Place the array membrane on a plastic sheet protector.

Pipette 1 mL of the prepared Chemi Reagent Mix evenly onto the membrane.

Cover the membrane with the top sheet of the plastic protector

Incubate for 1 minute.

Blot off excess Chemi Reagent Mix.

Wrap the membrane and sheet protector in plastic wrap.

Place the wrapped array membrane in an autoradiography film cassette and expose to X-ray film.

ARY027 Step 9

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FAQs

  • 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.

Product Datasheets

Certificate of Analysis

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