Cells were pulse‐labeled with 100 μCi/60 mm dish of EXPRE35S Protein Labelling Mix (PerkinElmer) for 30 min. Labeled cells were washed twice with ice‐cold PBS and chased in complete medium (DMEM containing 10% FBS) for 0–4 h. Pulse‐labeling and chase were also carried out in the presence and absence of 5 mM Azetidine‐2‐carboxylic acid (Azc). Pulse‐labeled cells were washed twice with PBS and incubated in lysis buffer (30 mM Tris–HCl pH 7.5, 150 mM NaCl and 1% Triton X‐100) for 30 min on ice and centrifuged at 10,000 g for 20 min at 4°C. After pre‐clearing using protein G Sepharose (GE Healthcare Bio‐Sciences), lysates were incubated overnight with anti‐AGR2 antibody (1:500) at 4°C. The beads were then added to the immune complexes and precipitated for 40 min at 4°C with gentle rotation and washed five times with lysis buffer. Immunoprecipitates were eluted with Laemmli sample buffer containing 50 mM DTT for 10 min at 70°C. The proteins were analyzed by both X‐ray fluorography and immunoblotting and detected using LumiGLO chemiluminescent substrate system (Kirkegaard & Perry Laboratories).
Lumiglo chemiluminescent substrate system
Manufactured by LGC
Sourced in United States
The LumiGLO chemiluminescent substrate system is a lab equipment product designed for the detection and visualization of proteins in Western blot analysis. It utilizes a chemiluminescent reaction to produce light, which can be captured and measured to quantify the presence and abundance of the target proteins.
Automatically generated - may contain errors
3 protocols using lumiglo chemiluminescent substrate system
1
Pulse-chase analysis of AGR2 protein
2
Western Blot Analysis of Autophagy Markers
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Proteins were extracted in lysis buffer (30 mM Tris, pH 7.5, 150 mM sodium chloride, 1 mM phenylmethanesulfonyl fluoride, 1 mM sodium orthovanadate, 1% Nonidet P-40, 10% glycerol, and phosphatase and protease inhibitors), separated by SDS-PAGE and electrophcoretically transferred onto polyvinylidene fluoride membranes. The membranes were probed with antibodies (LC3, PARP, ATG7 and β-actin) overnight at 4°C, and then incubated with a horse radish peroxidase-coupled secondary antibody. Detection was performed using a LumiGLO chemiluminescent substrate system [Kirkegaard & Perry Laboratories, Inc. (KPL), Gaithersburg, MD, USA].
3
Western Blot Analysis of Cellular Proteins
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After treatments, cells were placed on ice, washed twice with PBS, and lysed by boiling for 5-10mins in SDS sample buffer without reducing agent (50 mM Tris [pH 6.8], 2% sodium dodecyl sulfate, 10% glycerol) supplemented with Complete mini protease and phosphatase inhibitor cocktail tablets (Roche Diagnostics, and Pierce), 20 mM β-glycerophosphate, and 10 mM NaF for Western analysis. Lysates were centrifuged at 13,000 rpm for 15min at 4°C. Protein concentrations of each sample were determined using BCA protein assay kit as recommended by the manufacturer (Pierce, Hercules, CA). Proteins were resolved on sodium dodecyl sulfate–8% polyacrylamide gels and transferred onto Immun-Blot® PVDF Membrane with a wet transfer system (Bio-Rad). Membranes were blocked in blocking buffer (PBS containing 0.1% Tween 20 and 5% skim milk). Primary antibodies were used at indicated dilutions (Table S2). The blots were incubated in blocking buffer for 1h at room temperature in blocking buffer (for ATF6α) or overnight at 40C followed by incubations with secondary antibodies at room temperature for 1h. The proteins were then analyzed by immunoblotting and detected using LumiGLO chemiluminescent substrate system (Kirkegaard & Perry Laboratories) and visualized by exposure to autorad blue X-ray film (Research Products International).
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