Readyprep protein extraction kit total protein

Total proteins of fresh wood samples from five different time-points (0, 2, 6, 12, and 24 h) and from three biological replicates were extracted using the ReadyPrep™ Protein Extraction Kit (Total Protein) (Bio-RAD, USA) following the manufacturer's protocol. In total, 1 g of wood sample was powdered in liquid nitrogen using a mortar and pestle. The sample was transferred into a 5 mL microcentrifuge tube containing 2 mL of 2-D rehydration/sample buffer I, 20 μL of ReadyPrep TBP reducing agent and 0.2% (w/v) of the final concentrate of ampholyte. Subsequent steps were carried out according to the manufacturer's protocol. Total protein concentration was determined using the Quick Start™ Bradford protein assay kit (Bio-Rad, USA) on a Nanophotometer™ (IMPLEN, Germany) and bovine serum albumin (Bio-Rad, USA) was used as a standard. Upon quantification, 5 mg of total protein was transferred into a new 1.5 mL microcentrifuge tube, precipitated using 2-D Clean Up Kit (GE Healthcare Life Science, UK), following the manufacturer's protocol, and stored at −20 °C prior to in-solution tryptic digestion.

Proteins were extracted from cells (3×10⁴ cells/ml) using ReadyPrep™ Protein Extraction kit (Total Protein; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein concentration was measured with the bicinchoninic acid assay. Following denaturing, proteins (35 µg) were separated by 10% SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membrane by semi dry method. Membranes were blocked in 5% milk for 2 h at room temperature. Membranes were then incubated with the rabbit anti-human primary antibodies against HIF1α (1:1,500; cat. no. ab2185, Abcam, Shanghai, China) and GAPDH (1:2,000; cat. no. ab9485; Abcam) at 4°C overnignt, followed by secondary antibody horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:1,000; cat. no. MBS435036; MyBioSource, San Diego, CA, USA) at room temperature for 2 h. Signals were developed using ECL™ (Sigma-Aldrich; Merck KGaA) and scanned by MYECL™ Imager (Thermo Fisher Scientific, Inc.). Densitometric analysis was performed using ImageJ v1.46 software (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as endogenous control for data normalization.