Proteome Profiler Human Ubiquitin Array
Proteome Profiler Human Ubiquitin Array Summary
To detect changes in ubiquitination of 49 different proteins between samples. No specialized equipment is necessary
- Troubleshooting Guide
- Assays and antibodies for Analytes represented in the Human Ubiquitin Array Kit
General Assay Principle
Carefully selected capture antibodies have been spotted in duplicate on nitrocellulose membranes. Cell lysates are diluted and incubated with the Human Ubiquitin Array overnight. The array is washed to remove unbound proteins, followed by incubation with a biotinylated detection antibody. Streptavidin-HRP and chemiluminescent detection reagents are applied, and a signal is produced at each capture spot corresponding to the level of ubiquitinylation of the bound protein. Chemiluminescence is detected in the same manner as a Western blot.
- 4 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/Other Supplies Required sections of the product datasheet.
Stability and Storage
Reagents are stable for 12 months from date of receipt when stored in the dark at 2° C to 8° C.
| Simultaneously detect the Ubiquitination of these proteins in a single sample. | ||
| A20/TNFAIP3 | ATF4 | Bcl-2 |
| beta-TrCP1/BTRC | Caspase-8 | CBL |
| Cyclin D1/CCND1 | CD44 | cIAP-1/HIAP-2 |
| cIAP-2/HIAP-1 | COX-2/PTGS2 | EGF R |
| ER-alpha/NR3A1 | ErbB2 | ErbB3 |
| ErbB4 | FASN | F-box protein 15/FBXO15 |
| FBXW7/Cdc4 | FGF R2 | HGF R/c-met |
| HIF-1 alpha | HSP70 | HSP90AA1/HSP90a |
| IGF-I R | I kappa B alpha | I kappa B eplison |
| IKK gamma/NEMO | Insulin R | IRAK1 |
| IRF3 | IRS1 | M-CSF R |
| MSP R/Ron | Nrf2/NFE2L2 | NIK/MAP3K14 |
| p21CDKN1A/Cip1 | p53 | PDGF R alpha |
| PDGF R beta | RIP1 | SCF R/c-Kit |
| TfR | TNF R1/TNFRSF1A | TRAF-2 |
| TRAF-3 | TRAF-6 | Trk A |
| VEGF R3/Flt-4 | ||
Assays and antibodies for Analytes represented in the Human Ubiquitin Array Kit
Specifications
Product Datasheets
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
- 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)
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
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
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.
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Citations: Showing 1 - 5
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Targeting the Proteasome in Advanced Renal Cell Carcinoma: Complexity and Limitations of Patient-Individualized Preclinical Drug Discovery
Authors: J Li, L Pohl, J Schüler, N Korzeniews, P Reimold, A Kaczorowsk, W Hou, S Zschäbitz, C Nientiedt, D Jäger, M Hohenfelln, A Duensing, S Duensing
Biomedicines, 2021-05-31;9(6):. 2021-05-31
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The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells
Authors: S Hafner, M Schmiech, SJ Lang
Frontiers in Pharmacology, 2021-05-05;12(0):611657. 2021-05-05
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MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells
Authors: Q Wei, S Pinho, S Dong, H Pierce, H Li, F Nakahara, J Xu, C Xu, PE Boulais, D Zhang, M Maryanovic, AM Cuervo, PS Frenette
Nature Communications, 2021-05-04;12(1):2522. 2021-05-04
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Shifts in Ribosome Engagement Impact Key Gene Sets in Neurodevelopment and Ubiquitination in Rett Syndrome
Authors: DC Rodrigues, M Mufteev, RJ Weatheritt, U Djuric, KCH Ha, PJ Ross, W Wei, A Piekna, MA Sartori, L Byres, RSF Mok, K Zaslavsky, P Pasceri, P Diamandis, Q Morris, BJ Blencowe, J Ellis
Cell Rep, 2020-03-24;30(12):4179-4196.e11. 2020-03-24
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TRAF4-mediated ubiquitination of NGF receptor TrkA regulates prostate cancer metastasis
Authors: R Singh, D Karri, H Shen, J Shao, S Dasgupta, S Huang, DP Edwards, MM Ittmann, BW O'Malley, P Yi
J. Clin. Invest., 2018-06-18;0(0):. 2018-06-18
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