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Proteome Profiler Human Ubiquitin Array
R&D Systems, Inc. a Bio-Techne Brand
Catalog # ARY027
Key Product Details
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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
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.
Place each array membrane in a separate well of the 4-Well Multi-dish.
Incubate for one hour on a rocking platform shaker.
Adjust volume of each sample to final volume of 1.5 mL with Array Buffer 1.
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.
Add 15 µL of Detection Antibody to 1.5 mL of Array Buffer 2 for each array.
Pipette 1.5 mL diluted Detection Antibody Cocktail into each well of the 4-Well Multi-dish.
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.
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.
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.
Citations for Proteome Profiler Human Ubiquitin Array (5)
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Citations are publications that use Bio-Techne products. Selected citations for Proteome Profiler Human Ubiquitin Array include:
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Species: Human
Sample Types: Whole Cells
J Li et al. (2021), Targeting the Proteasome in Advanced Renal Cell Carcinoma: Complexity and Limitations of Patient-Individualized Preclinical Drug Discovery Biomedicines, 9(6).
PMID: 34072926 -
Species: Mouse
Sample Types: Cell Lysates
Q Wei et al. (2021), MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells Nature Communications, 12(1):2522.
PMID: 33947846 -
Species: Human
Sample Types: Cell Lysates
S Hafner et al. (2021), The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells Frontiers in Pharmacology, 12(0):611657.
PMID: 34025398 -
Species: Human
Sample Types: Cell Lysates
DC Rodrigues et al. (2020), Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome Cell Rep, 30(12):4179-4196.e11.
PMID: 32209477 -
Species: Human
Sample Types: Protein
R Singh et al. (2018), TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis J. Clin. Invest., 0(0).
PMID: 29715200
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FAQs for Proteome Profiler Human Ubiquitin Array
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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.
