Cell lysates were created using RIPA buffer (Boston Bio-Products; MA) supplemented with protease and phosphatase inhibitors (Thermo Scientific; MA). Briefly, the sample proteins (30 or 40 μg) were separated by SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore; MA). After blocking with 5% BSA or non-fat milk for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The next day, the membranes were incubated with an anti-rabbit or mouse secondary antibody (Bio-Rad; CA) for 1 h. Finally, the proteins were detected using ECL Plus Western Blotting Detection Reagents (GE Healthcare; PA). The cytokine/growth factor array analysis kit (AAH-GF-1) was purchased from RayBiotech The following primary antibodies were used for immunoblot: anti-KIBRA (# 8774), anti-phospho-YAP1 (S127) (#13008), anti-AKT (# 4685), anti-phospho-AKT (# 4060), anti-ERK (# 4695) and anti-phospho-ERK (# 9101) from Cell Signaling Technology, MA; anti-AREG (16036-1-AP) from Proteintech, IL; anti-YAP1 (SC-15407) from Santa Cruz Biotechnology, CA; anti-GAPDH (Y1041) and β-actin (Y1051) from Ubiquitin-Proteasome Biotechnologies, CO.
Aah gf 1
Manufactured by RayBiotech
Sourced in United States
The AAH-GF-1 is a lab equipment product designed for general laboratory use. It serves as a tool for various applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and purely factual approach. This product's specific capabilities and intended uses are not available from the information provided.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Microsoft excel based analysis software tools, by RayBiotech (1 mentions)
4 protocols using aah gf 1
1
Western Blot Analysis of Cellular Signaling
2
Growth Factor Profiling in PPP and ePRP
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Platelet poor plasma (PPP) and ePRP were assayed for the presence of human growth factors using a cytokine array (Cat. AAH-GF-1, RayBiotech, Peachtree Corners, GA, USA) and performed according to the manufacturer’s instructions. The data analysis has been employed by RayBiotech Microsoft® Excel-based Analysis Software Tools for automated analysis (N = 40 individual human growth factor cytokines were detected).
3
Growth Factor Profiling of ECCM
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The growth factor array AAH-GF-1 (RayBiotech) was performed on 3 independently derived ECCM samples and 1 control sample (CSC medium -GFs) following the manufacturer’s instructions. Detection was carried out using the LAS4000 and the spot intensity was quantified using the Odyssey V3.0 program. From each value of the ECCM the value of the control medium was subtracted and the average of the duplicates was calculated. Displayed in the graph are the raw intensities of the 3 different ECCM samples taken together.
4
Evaluating Secreted Growth Factors from Mesenchymal Cells
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The human growth factor array (cat# AAH-GF-1, RayBiotech, Inc., Noncross, GA, USA) was used to evaluate the growth factors secreted from MSCs or DPCs. The DPCs were plated in 60-mm culture dishes at 2 × 105 cells and incubated for 24 h. They were then cultured for 48 h with 50% P-CM in FBS-free DMEM medium, and the culture supernatants were collected. A cytokine antibody array was conducted according to the manufacturer’s protocol (Supplementary Figure S2 ). The membranes were incubated in blocking buffer at room temperature (RT), and the membranes were then incubated with 1 mL supernatants collected from P-CM or P-CM-treated DPCs for 4 h at RT. The membrane was washed and then incubated with a biotinylated antibody cocktail. The antibodies bound to the array were detected using HRP-streptavidin and an enhanced chemiluminescence detection system, and the spot intensity was quantified using densitometry on Image J software.
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