Raybio aam blm 1 label based mouse antibody arrays

To assess similarities and differences in soluble factors present under different LCM conditions, RayBio AAM-BLM-1 label-based mouse antibody arrays were used to simultaneously assess the expression of 308 soluble murine target proteins (RayBiotech Inc, Norcross, GA, USA). Post-dialysis protein concentration of LCM (n = 3 per condition) was determined using the DC protein assay (Bio-Rad Laboratories, Mississauga, ON, Canada), labeled and incubated with protein arrays as per manufacturer’s instructions. Results were visualized using chemiluminescence and film exposure (CL-XPosure Film; Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Results (n = 3 per media condition) were analyzed using the RayBiotech analysis tool for AAM-BLM-1. Sixteen confirmed protein hits unique to LCM generated from the lungs of mice bearing SUM159 tumors were identified as having values > 1 after background subtraction and validation across three replicates. Due to differences in antibody affinities for target antigens, quantitative comparison between different proteins was not feasible using this platform.

To identify soluble factors present within BMCM, RayBio® AAM-BLM-1 label-based mouse antibody arrays were used to simultaneously assess of expression of 308 soluble murine target proteins (RayBiotech Inc., Norcross, GA) as described previously [20 (link)]. Results were visualized using chemiluminescence and film exposure (CL-Xposure Film, Pierce). Densitometric analysis was conducted using Image J with the MicroArray Profile Macro and results (N = 3/media condition) were analyzed using the RayBiotech analysis tool for AAM-BLM-1 as described previously [20 (link)]. To identify potential phosphorylation changes in human breast cancer cells in response to BMCM, MDA-MB-231 human cells were incubated in basal media, BMCM, or BMCM depleted of OPN and cell lysates were harvested after 2 hours. Protein concentrations were determined with a DC protein assay (BioRad). Cell lysates were incubated with the Human Phospho-Kinase Array membranes (ARY003B, R&D Systems) overnight at 4°C. Biotinylated detection antibodies were applied and membranes were visualized using chemiluminescence. Densiotometric analysis was performed using the Protein Array Analyzer for ImageJ (N = 3/ media condition).