For each group, three rat L4–L5 spinal cord segments were selected and stored in liquid nitrogen; 600 mL of RIPA lysis solution (P0013C, Beyotime, China) was added for total protein extraction. After extracting proteins from rat spinal cord tissue, the protein concentration was adjusted to 1 μg/μL using the SDS-PAGE protein sample loading buffer (B1012, Applygen, China). Protein samples (10 μg) were separated by SDS-PAGE and transferred to PVDF membranes (R1DB92455, Millipore, Germany), which were then incubated with primary antibodies at 4 °C overnight. The primary antibodies used were toll-like receptor 4 (TLR4) (1:1000, ab13556, Abcam), IKBα (1:1000, ab12134, Abcam), p-IKBα (1:1000, ab133462, Abcam), and α-tubulin (1:5000, ab7291, Abcam). The electroporated membrane was washed with TBST, incubated with the secondary antibody for 1 h at room temperature, and developed. Quantitative analysis of the protein bands was performed using ImageJ software. All western blots represent data from three independent replicates.
B1012
Manufactured by Applygen
Sourced in China
The B1012 is a compact and versatile laboratory equipment designed for general scientific applications. It features precise temperature control and stable performance to support a wide range of laboratory experiments and procedures.
Automatically generated - may contain errors
2 protocols using b1012
1
Quantification of Spinal Cord Proteins
2
Western Blot Analysis for Protein Quantification
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The PAG, RVM, TNC, and colon specimens were homogenized in PIRA lysis buffer (WB3100, NCM, China) containing 1% protease inhibitor cocktail (p001, NCM, China). Then, the homogenates were centrifuged at 12000 rpm for 20 min. The supernatant of dissolved proteins was collected and determined by BCA (bicinchoninic acid) kits (WB6501, NCM, China). The loading buffer (B1012, Applygen, China) was added to the unified proteins' supernatant in a 1 : 4 ratio. Proteins were separated in 8%, 10%, or 12% gradient gels via sodium and transferred to polyvinylidene fluoride membranes (PVDF). The nonspecific binding sites on PVDF were blocked with 5% nonfat milk (P1622, Applygen, China) for 2 h, and the membranes were washed 3 times and placed in primary antibodies overnight at 4°C. The molecular weight, No., brand, and dilution ratio of all primary antibodies were shown in Supplementary 2 . Then, the membranes were washed 3 times and incubated with the horseradish peroxidase-conjugated secondary antibodies (SA00001-1 and SA00001-2, 1 : 10000, Proteintech, America; sc-2789, 1 : 10000, Santa Cruz, America) for 1 h at room temperature. The membranes were washed again for 3 times. The gel imaging system (Fusion, Germany) was used to detect antibody-reactive bands with chemiluminescence reagent kit (P10200, NCM, China), which were analyzed by ImageJ software.
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