Medium 200 (M200PRF500), low serum growth supplement (LSGS; S00310), 0.025% trypsin-EDTA (R001100), VEGF (PHC9344), Pierce Protease Inhibitor Mini Tablets (A32953), HisPur™ Cobalt Resin (89964), Detoxi-Gel™ (20339), penicillin-streptomycin (15140122), amphotericin B (15290018), Qubit™ Protein Broad Range kit (A50668) and human umbilical vein endothelial cells (HUVECs; C0035C; RRID: CVCL K312 ) were purchased from Thermo Fisher Scientific/Fisher Scientific. ToxinSensor™ LAL Endotoxin Assay Kit (L00350) was purchased from Genscript. HisLink™ Protein Purification Resin (V8823) and CellTiter 96® AQueous One Solution (G3582) were purchased from Promega. Mitomycin C (M4287) was purchased from Sigma Aldrich. Proteome Profiler™ Human Angiogenesis Antibody Array (ARY007) was purchased from R&D Systems. IRDye 800 CW Streptavidin (926-32230) was purchased from LI-COR. Growth factor reduced Matrigel (GFR-Matrigel; 356231) was purchased from Corning. Axitinib (HY-10065) was purchased from MedChemExpress. 0.1 mm glass beads (P000929LYSK0A.0) and soft tissue homogenizing kits (P000933-LYSK0-A) were purchased from Bertin Corp. 4-well culture inserts (80466) and angiogenesis slides (81506) were purchased from ibidi. MycoAlert™ Plus Mycoplasma Detection Kit (LT07-701) was purchased from Lonza.
Proteome profiler human angiogenesis antibody array
Manufactured by R&D Systems
Sourced in United States
The Proteome Profiler™ Human Angiogenesis Antibody Array is a multiplex assay that allows for the simultaneous detection and quantification of 55 different angiogenesis-related proteins in a single experiment. The array utilizes capture antibodies spotted in a 4 x 15 grid format on a membrane to capture target analytes from a sample. Detection is achieved through the use of a cocktail of biotinylated detection antibodies and a chemiluminescent readout.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Slide angiogenesis, by Ibidi (1 mentions)
Axitinib, by MedChemExpress (1 mentions)
Hislink protein purification resin, by Promega (1 mentions)
Irdye 800cw streptavidin, by LI COR (1 mentions)
Detoxi gel, by Thermo Fisher Scientific (1 mentions)
4 well culture inserts, by Ibidi (1 mentions)
Hispur cobalt resin, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Mycoalert plus mycoplasma detection kit, by Lonza (1 mentions)
Celltiter 96 aqueous one solution, by Promega (1 mentions)
Vivaspin concentrator, by Sartorius (1 mentions)
Fluorchem sp, by Bio-Techne (1 mentions)
Proteome profiler human cell stress array, by R&D Systems (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Image studio acquisition software, by LI COR (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
14 protocols using proteome profiler human angiogenesis antibody array
1
HUVEC Culture and Angiogenesis Assays
2
Quantifying HUVEC Angiogenesis Factors
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At least 24 h after seeding, culture medium of HUVECs was refreshed with or without 25 μM CQ. Forty-eight hours later, HUVECs established confluent monolayer. Conditioned culture medium was harvested and centrifuged to deplete floating cells and cell debris. Conditioned HUVEC culture supernatants were analyzed using a Proteome Profiler Human Angiogenesis Antibody array (R&D systems, according to manufacturer's manual) and chemiluminescence for detection. Densitometry was done with Image Lab software. HUVECs were also lysed and protein abundance was assessed to correct densitometry values.
3
Profiling Angiogenic Factors in MSC-Derived Secretome
4
Profiling Angiogenesis-related Proteins
5
Angiogenesis Antibody Array Protocol
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Proteome Profiler Human Angiogenesis Antibody Array (Catalog # ARY007, R&D Systems) was used for the angiogenesis related factors detection according to the manufacturer's instruction. Briefly, CM was first mixed with the detection antibody cocktail at room temperature for 1 h prior to being added to the array membrane. The membrane was then incubated overnight at 2–8°C on a shaker. After washing, horseradish peroxidase–conjugated streptavidin was next added to the membrane followed by 30 minutes incubation at room temperature on a shaker. After washing, X-ray film and a chemiluminescence imaging system were used to detect and quantify the array signals.
6
Profiling Angiogenesis-related Proteins
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The relative expression levels of 55 angiogenesis-related proteins were detected in the cell culture supernatant using a Proteome Profiler™ Human Angiogenesis Antibody Array according to the manufacturer’s instructions (R&D Systems, cat. no. ARY007, Minneapolis, MN, USA). Briefly, after the 1-h membrane blocking step, cell culture supernatant (200 µL) was pre-incubated for 1 h with biotinylated detection antibodies cocktail (15 µL) and then added to the membrane before incubating overnight at 4 °C on a rocking platform shaker. After a series of washing steps, the membrane was incubated with 2 mL of diluted streptavidin-horseradish peroxidase for 30 min before chemiluminescence detection on a Fusion SL system (PeqLab/VWR, Langenfeld, Germany).
7
Characterizing MSC Secretome Angiogenic Factors
8
Profiling Angiogenesis-Related Proteins in Cell Cultures
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Proteome Profiler Human Angiogenesis Antibody Array (R&D Systems, Inc., Minneapolis, MN, USA) was used to measure the relative expressions of angiogenesis-related proteins. Briefly, conditioned medium was collected from SDC-1 over-expressing, SDC-1 silenced cells, and their corresponding controls; i.e., EGFP and negative siRNA, respectively. The conditioned medium was mixed with a cocktail of biotinylated detection antibodies and added to membranes coated with primary antibodies against 55 angiogenesis-related proteins and incubated overnight at 4 °C. Then, membranes were incubated for 30 min with streptavidin-horseradish peroxidase (HRP) and chemiluminescence detection reagents were added for approximately 1 min. Dot blots were registered with CCD camera (FluorChem SP, Alpha Innotech, San Leandro, CA, USA). The average pixel density of duplicate spots on the membrane was determined using ImageJ software (http://rsb.info.nih.gov/ij/ ).
9
Quantifying Angiogenic and Cell Stress Factors
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Cell culture supernatants and lysates were centrifuged at 500 g for 5 min, and aliquots were stored at − 80 C until further use. Samples from two biologically distinct experiments were pooled for analysis, and each experiment was conducted using 3 technical replicates of samples. Angiogenic factors were measured in the cell supernatant using the Proteome Profiler™ Human Angiogenesis Antibody Array, and cell stress markers were measured in cell lysates using the Proteome Profiler™ Human Cell Stress Array (R&D system, USA) according to the manufacturer’s protocols. Briefly, 500 ml of supernatant or 150 mg of cell lysate was incubated with a cocktail of biotinylated detection antibodies and added to the array membrane. Following incubation on a rocker at 4 °C overnight, capture antibodies spotted in duplicate on the array were visualized using chemiluminescent detection reagents. The signal produced by each spot is proportional to the amount of bound analyte. The mean pixel intensity of the duplicate spots on the membrane was calculated and averaged using ImageJ Software.
10
Angiogenic Factors Profiling in Tumors
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Upregulation of angiogenesis-related factors was determined using a Proteome Profiler™ Human Angiogenesis Antibody Array (R&D Systems, Minneapolis, MN, USA, cat. no. ARY007), which allows the identification of angiogenic factors present in tumour lysates. The array consists 59 types of nitrocellulose membrane-bound antibodies specific for various angiogenic factors. The array was conducted according to the manufacturer’s protocol. Briefly, tumour tissue extracts were prepared from xenografted tumours derived from the SK-MEL-28 and SK-IL13Rα2 cells and incubated with a cocktail of biotinylated antibodies against various angiogenic factors. The mixture was then incubated with membrane-bound anti-angiogenic factors antibodies, followed by incubation of trapped complexes with HRP-conjugated streptavidin, and detection with chemiluminescent detection reagent.
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