Proteome Profiler Human Ubiquitin Array ARY027: R&D Systems
Kit Summary

To detect changes in ubiquitinylation of 49 different proteins between samples. No specialized equipment is necessary

 

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.

 

Kit Contents
  • 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 Ubiquitin  

 

Assays and antibodies for Analytes represented in the Human Ubiquitin Array Kit


Analyte
Affinity Purified Polyclonal Antibodies
Monoclonal Antibodies
Labeled Antibodies Quantikine, DuoSet, or DuoSet IC ELISA
A20/TNFAIP3   MAB7598    
ATF4   MAB7218    
Bcl-2 AF810 MAB8272, 2291-MC-100, MAB827   DYC827B
beta-TrCP1/BTRC   MAB7675    
Caspase-8 AF1650, AF705 MAB704, MAB8135    
CBL AF5998 MAB5998    
Cyclin D1/CCND1 AF4196      
CD44 AF3660 MAB7045, BBA10, BBA13   DY7045
cIAP-1/HIAP-2 AF8181 MAB818    
cIAP-2/HIAP-1 AF8171 MAB817 BAF8171  
COX-2/PTGS2 AF4198 MAB4198   DYC4198
EGF R AF231 MAB1095 BAF231 DEGFR0, DY231, DYC1854
ER-alpha/NR3A1 AF5715 MAB57151, PP-H4624-00   DYC5715
ErbB2 AF1129 MAB1129 BAF1129 DHER20, DY1129, DYC1129
ErbB3 AF234 MAB3482, MAB348 BAF234 DY348, DYC234
ErbB4 AF1131 MAB1131 BAF1131 DYC1133
FASN AF5927 MAB59271, MAB5927    
F-box protein 15/FBXO15 AF6035      
FBXW7/Cdc4   MAB7776    
FGF R2   MAB6842, MAB684   DYC665
HGF R/c-met AF276 MAB5694 BAF358 DY358, DYC358
HIF-1 alpha AF1935 MAB1536   DYC1935
HSP70 AF1663 MAB1663   DY1663, DYC1663
HSP90AA1/HSP90 alpha AF3775, AF3286, AF7247 MAB3286    
IGF-I R AF-305-NA MAB391 BAF391 DY391, DYC305
IκB alpha AF4299 MAB4299   DYC4299
IκB epsilon AF4300 MAB4300    
IKK gamma/NEMO AF2684, AF4365      
Insulin R AF1544 MAB15441 BAF1544 DYC1544
IRAK1 AF4048      
IRF3 AF4019 MAB4019    
IRS1 AF3978 MAB39782    
M-CSF R AF329 MAB3291, MAB329 BAF329 DY329
MSP R/Ron AF691   BAF691 DYC691
Nrf2/NFE2L2 AF3925 MAB3925    
NIK/MAP3K14        
p21CDKN1A/Cip1 AF1047 MAB1047 BAF1047 DYC1047
p53 AF1355 MAB1355 BAF1355, HAF1355 DYC1043
PDGF R alpha AF-307-NA MAB322 BAF322 DYC322
PDGF R beta AF385 MAB1263, MAB385 BAF385 DYC385
RIP1   MAB3585    
SCF R/c-Kit AF332, AF3267 MAB332 BAF332 DSCR00, DY332
TfR AF2474   BAF2474 DTFR1, DY2474
TNF R1/TNFRSF1A AF225, AB-225-PB MAB625, MAB225 BAF225 DRT100, DY225
TRAF-2 AF3277 MAB3277    
TRAF-3 AF3278 MAB3278    
TRAF-6 AF3284      
Trk A AF175 MAB175   DYC175
VEGF R3/Flt-4 AF349 MAB3491 BAF349 DY349, DYC3491
Ubiquitin A-101, A-100 MAB701, A-106    
Preparation and Storage
  • Stability & Storage
    Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Related Research Areas
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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