Antibody array assay kit

OPCs were plated at 1 × 10⁶ cells per well in a 6-well PDL-coated plate and treated with activin-A (10 ng ml⁻¹) or vehicle control (0.0002% BSA). Cultures were washed with cold phosphate buffered saline on ice, scraped, and centrifuged thrice at 10,000 rpm at 4 °C and supernatant discarded. Protein extraction, lysate purification, and protein biotinylation were performed using the Antibody Array Assay Kit (Full Moon Biosystems) according to the manufacturer’s instructions. Samples were applied to TGF-β phospho-antibody microarray slides (Full Moon Biosystems) which have 176 immobilized antibodies against phosphorylated and unphosphorylated specific residues in proteins associated with the 5 TGFβ signaling pathways, with 6 technical replicates per antibody. All antibodies are against activating phosphorylation states, to the exception of Abl Thr754/735, GEF2 Ser885, Cofilin Ser3, Myc Ser373/Ser62/Thr358/Thr58. Following incubation with streptavidin-Cy3 (1:1000), signal was detected on an Axon4200 microarray scanner (Edinburgh Genomics, The University of Edinburgh). Subsequent to background signal subtraction, values from phosphorylated signal were normalized to total protein signal for each protein site, then normalized to vehicle control. Data were then Log2 transformed and plotted as heat maps using GraphPad Prism 7 (San Diego, USA).

Antibody arrays were purchased from Full Moon BioSystems (PMK185 and PEG214). Biotinylation of the proteins and conjugation and detection by Cy3-streptavidin (PA43001; GE Healthcare Life Science, Little Chalfont, UK) were performed using an antibody array assay kit (Full Moon BioSystems) according to the manufacturer’s instructions. Protein samples (60 μg) were used in the antibody array assay. To calculate the STR using Eq. (5), the detection assay was performed six times independently following the addition of EGF to the cultures (50 ng/mL using the PEG214 array and 100 ng/mL using the PMK185 array). The mean fluorescent intensity (± standard deviation) was utilized to calculate the transduction characteristics. The antibody arrays were scanned using a SureScan Microarray Scanner (G2565CA Microarray Scanner System; Agilent Technologies, Santa Clara, CA, USA), after which the acquired image data were analyzed. The signal intensity was normalized by dividing the result by the negative control values from the array. Each value for phosphorylated proteins was divided by the respective value for unphosphorylated proteins at 0, 15, 30, 45, 60, 120, and 180 min. Finally, the results were divided by the value at 0 min to calculate the increase in phosphorylated molecules. The coefficient of variation for six replicates was < 0.1^{33 (link)}.

The antibody array assay kit was procured from Full Moon BioSystems (Sunnyvale, CA, USA). This technique was used as an alternative to procuring phospho-specific antibodies individually and performing several western blot assays. The protocol was carried out as per manufacturer’s instructions with the following alteration to the homogenization step: instead of using the bead and vortex homogenization indicated in the kit, the hand-held Qiagen TissueRuptor was used.

The anti-phosphoprotein microarray PGP193, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA), contains 193 antibodies. Each of the antibodies has six replicates that are printed on coated glass microscope slides, along with multiple positive and negative controls. In brief, cell lysates obtained from HEC-1B/mock and HEC-1B/shAMF-1 were biotinylated with the Antibody Array Assay Kit (Full Moon Biosystems, Inc.). The antibody microarray slides were first blocked in a blocking solution (Full Moon Biosystems, Inc.) and dried with compressed nitrogen. The slides were then incubated with the biotin-labeled cell lysates (∼100 μg protein) in coupling solution at room temperature for 2 h and rinsed extensively with Milli-Q grade water before detection of bound biotinylated proteins using Cy3-conjugated streptavidin. The slides were scanned on a GenePix 4000 scanner and the images were analyzed with GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA). The fluorescence signal for each antibody was obtained from the fluorescence intensity of this antibody spot. A ratio computation was used to assess the extent of protein phosphorylation. For each antibody that has phosphorylated and matching unphophorylated values in both the control data and experiment data are represented as “phospho” and “unphospho”.

The JAK-STAT-protein profiling was performed using the Jak/Stat Phospho Antibody Array (PJS042) and the antibody Array Assay Kit (KAS02), designed and manufactured by Full Moon Biosystems (Sunnyvale, CA, USA). The proteins microarray analysis was carried out according to the manufacturer's instructions. Briefly, 5X10⁶ BMK-16/myc cells were cultivated on cell culture flasks and were treated with 100 ng/ml of ovine IFN-τ during 15 min; a non-treated cell culture was used as control. After the cells were harvested using a scraper and lysed (with Lysis beads and buffer), the clarified-protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, Ill, USA). One hundred micrograms of cell lysate was labeled with biotin in DMF (10 μg/μl) (N,N-Dimethylformamide), and biotin-labeled were diluted 1:20 in a coupling solution before applying them to the array for conjugations. The antibody microarray was blocked before the incubation with the biotin-labeled cell lysates at room temperature (RT) during 4 hrs. Then, the slides were washed; subsequently, 60 μ of Cy3-streptavidin in detection buffer (0.5mg/ml) were added, and the mix was incubated for 20 min at RT. After the slides were washed with Milli-Q grade water, they were dried by centrifugation before the fluorescence detection.