Protein Extraction and Western Blotting

Total protein was extracted using RIPA kit (AS1004, ASPEN), and protease inhibitors (AS1008, ASPEN) were added during protein extraction to prevent protein degradation. After electrophoresis, coating and sealing, the indexes to be detected were incubated with primary antibody and secondary antibody, and finally immunolabeled with enhanced chemiluminescence reagent (AS1059, ASPEN). Antibodies against caspase-3 (#9662), caspase-12 (#9671), phosphorylated PERK (p-PERK, Thr980, #3179), PERK (#3192), phosphorylated eIF2α (p-eIF2α, Ser51, #3597), eIF2α (#2103), GRP78 (#3183), CHOP (#2895), and HO-1 (#43966) were obtained from cell signing technology corporation (Massachusetts, USA). Anti-β-tubulin (#ab6046), anti-β-actin (#ab8226), and anti-Nrf-2 (#ab137550) were purchased from Abcam (Cambridge, USA). Antihistone H3 (EM1108) was purchased from ELK Biotechnology (Wuhan, China).

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Lu X., Xu G., Lin Z., Song J., Zhang Y., Wang H., Lu F., Xia X., Ma X., Zou F, & Jiang J. (2023). Sulforaphane Delays Intervertebral Disc Degeneration by Alleviating Endoplasmic Reticulum Stress in Nucleus Pulposus Cells via Activating Nrf-2/HO-1. Oxidative Medicine and Cellular Longevity, 2023, 3626091.

Publication 2023

AS1004 AS1008 AS1059 Anti-β-tubulin (#ab6046), ab137550

Actin Antibodies Antibody Caspase 12 Caspase 3 Cell Chemiluminescence Chop Electrophoresis Grp78 Nrf 2 Protease inhibitors Protein Protein degradation Ripa Tubulin

Corresponding Organization : Fudan University

Top 5 similar protocols

Variable analysis

independent variables

Protein extraction method (RIPA kit)
Addition of protease inhibitors during protein extraction

dependent variables

Expression levels of caspase-3
Expression levels of caspase-12
Phosphorylation levels of PERK
Expression levels of PERK
Phosphorylation levels of eIF2α
Expression levels of eIF2α
Expression levels of GRP78
Expression levels of CHOP
Expression levels of HO-1
Expression levels of β-tubulin
Expression levels of β-actin
Expression levels of Nrf-2
Expression levels of histone H3

control variables

Electrophoresis conditions
Coating and sealing conditions
Incubation with primary and secondary antibodies
Immunolabeling with enhanced chemiluminescence reagent

positive controls

Not specified

negative controls

Not specified

Annotations

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