Total protein samples were extracted by treating tissues with RIPA lysis buffer (Biosharp, China). Protein concentration was determined using a Pierce BCA protein detection kit (Beyotime Institute of Biotechnology, Shanghai, China). The protein was separated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking in 5% skimmed milk, it was incubated with the primary antibodies occludin (Proteintech, 27260-1-AP, 1:1000), cyclooxygenase-2 (COX-2) (Proteintech, 66351-1-Ig, 1:1000), BDNF (Proteintech, 28205-1-AP, 1:1000), vascular cell adhesion molecule (VCAM) (Huabio, SA05-04, 1:1000), TLR4 (Bioworld, BS3489, 1:1000), Myd88 (Bioworld, BS3521, 1:1000), phospho c-Jun N-terminal kinase (p-JNK) (Proteintech, 80024-1-RR, 1:1000), JNK (Proteintech, 24164-1-AP, 1:1000), caspase-3 (Cell Signaling Technology, 9662S, 1:1000), and β-Tubulin (Proteintech, 10068-1-AP, 1: 50,000) at 4°C overnight. After washing with TBST three times, the bands were incubated with appropriate antirabbit or antimouse secondary antibodies at room temperature for 1 h. The imprinting was observed via chemiluminescence (Pierce) and an Odyssey imaging system (Li-Cor-Biosciences, NE).
Pierce bca protein detection kit
Manufactured by Beyotime
The Pierce BCA protein detection kit is a colorimetric assay used for the quantification of total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction, where proteins reduce Cu2+ to Cu+ in an alkaline environment, and the resulting Cu+ ions chelate with BCA to produce a purple-colored complex that can be measured spectrophotometrically.
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2 protocols using pierce bca protein detection kit
1
Protein Quantification and Western Blot Analysis
2
Isolation and Characterization of Nuclear Membrane Vesicles
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The frozen neutrophils were homogenized after thawing. The homogenate was extracted, centrifuged (3,500 rpm, 5 min) at 4°C, and the nuclei and intact cells were removed. The supernatant was centrifuged (20,000 rpm, 25 min) at 4°C to remove the precipitate and mitochondria. The supernatant was then centrifuged (50,000 rpm, 1 h) at 4°C. The NM vesicles containing precipitation were collected and washed with 0.2 mM EDTA Na2 twice. The NM vesicles were extruded on 400-nm and 100-nm polycarbonate membranes for several times continuously. After 5 min of ultrasound, the membranes were stored at −80°C for later use. The membrane content was determined by Pierce BCA protein detection kit (Beyotime Biotechnology).
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