Secondary goat anti rabbit igg hrp

Protein samples were diluted in 4X LDS Sample Buffer and 10X Reducing Buffer (Life Technologies, Carlsbad, CA, USA) to provide the given concentrations. Samples were then loaded on Bolt 4–12% Bis-Tris gels (Life Technologies) and run at 200 V for 35 min. Samples were transferred to nitrocellulose membranes and total protein loading was determined by Ponceau S staining. Samples were blocked with 3% BSA and incubated with either primary rabbit polyclonal anti-human sEH antibody (1:5000) (Yu et al. 2004), rabbit polyclonal anti-human EH1 antibody (1:5000) (Thermo Scientific, Waltham, MA, USA, Lot # QD2009572) or rabbit polyclonal anti-human EH3 antibody (1:5000) (MyBioSource, San Diego, CA, USA, Lot# MBS851755) and subsequently incubated with secondary goat anti-rabbit IgG-HRP (1:5000) (Abcam, Cambridge, MA, USA). Membranes were developed with SuperSignal West Femto ECL Detection Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and imaged with ChemiDoc MP (Bio-Rad Laboratories, Hercules, CA, USA). Quantification was performed from quantifying band intensity using ImageLab 5.0 (Bio-Rad). Values are given as the average ± standard deviation of at least 3 independent gels.

The osteogenic effect of the ASC and PSC was further tested in dMBMS cells using the western blot method, as an empirical study. In brief, the cells were seeded at a cell density of 1 × 10⁵ in 6 well plates and cultured with 1 mL culture medium for control. For collagen treatment, the samples (50 ng/mL) were mixed with 1 mL cultured medium and cultured for three days. Then, the treated and untreated cells were harvested using a lysis buffer with 30 s sonication and centrifuged at 12,000 rpm at 4 °C for 15 min for the extraction of total cellular protein. The protein content was quantified using a BCA kit as per the manufacturer’s instructions (Thermo Fisher Scientific, Shanghai, China) and then was separated by 12% SDS-PAGE and transferred to PVDF nitrocellulose membrane (Invitrogen, Shanghai, China) using iblot-2 dry blotting system (Invitrogen, Shanghai, China). A protein transferred membrane blocked with 5% BSA-PBST were incubated with primary antibodies such as anti-GAPDH, anti-Col1α2 and anti-Runx2 (ABcam, Shanghai, China) overnight at 4 °C. Then the membrane was incubated with secondary goat anti-rabbit IgG-HRP (Abcam) for 1 h at 37 °C and exposed to an enhanced chemiluminescent reagent. Images were captured with a Universal Hood II Gel Doc System (Bio-Rad, Rochester, NY, USA).

Fresh Arabidopsis leaves (0.1 g) were homogenized in 0.2 ml of protein extraction buffer (0.125 mM Tris, pH6.8, 4% w/v SDS, 18% glycerol, 0.024% w/v bromophenol-blue, 1.43 M β-mercaptoethanol, 0.2% protease inhibitor). After boiling for 10 min, the insoluble fraction was removed by centrifugation, and the supernatant (denatured protein) was separated on a 12% SDS PAGE gel and transferred onto a nitrocellulose membrane, followed by incubation with primary anti-GFP antibody (Abcam, ab290, Cambridge, MA, USA) and secondary goat anti-rabbit IgG HRP (Abcam) antibody. The membrane was developed with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The expression of SPL10-GFP fusion protein was also investigated using a Biological Confocal Laser Scanning Microscope FV10-ASW, and the emission wavelength for GFP and DAPI channels are 488 nm and 405 nm, respectively (OLYMPUS, Tokyo, Japan).