Quantitative real‐time PCR and western blot were conducted as our previously described.37 , 38 , 39 After being extracted by the TRIzol reagent, total RNA was converted to cDNA using oligo dT primers that served as a template to assess the relative gene expression on the Roche LightCycler® 480 detection system using SYBR green. Proteins were extracted from ventricules or cells and the concentrations were determined by a BCA kit. Fifty micrograms of the proteins were exposed to SDS‐PAGE (30% acrylamide) separation and then transferred onto PVDF membranes. After the incubation with indicating primary antibodies (4°C, overnight) and secondary antibodies (room temperature, 1 h), respectively, the membranes were then visualized by a ChemiDoc™ XRS+ chemiluminescence system and analyzed using an Image Lab Software (Bio‐Rad Laboratories, Inc.).
Chemidoc xrs chemiluminescence system
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc XRS chemiluminescence system is a laboratory instrument designed for the detection and analysis of chemiluminescence-based assays. It provides a versatile platform for imaging and quantifying a wide range of chemiluminescent samples, including Western blots, ELISA plates, and other chemiluminescent-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Solarbio (2 mentions)
Lightcycler, by Roche (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Protran ba83, by Cytiva (1 mentions)
Supersignal west dura extended substrate, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Protran, by Cytiva (1 mentions)
Hrp conjugated goat anti mouse igg, by Proteintech (1 mentions)
Hrp conjugated goat anti rabbit igg, by Proteintech (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti gapdh ab, by Proteintech (1 mentions)
Anti s100β, by Proteintech (1 mentions)
Anti cd31, by Proteintech (1 mentions)
Anti rhoa, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Solarbio (1 mentions)
Nitrocellulose nc membrane, by Pall Corporation (1 mentions)
Anti α sma, by Proteintech (1 mentions)
7 protocols using chemidoc xrs chemiluminescence system
1
Quantitative Real-Time PCR and Western Blot Analysis
2
Photodynamic Therapy Enhances Apoptosis in Cancer Cells
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HeLa cells were incubated with cell culture medium containing PBS, SCPB NPs, DOX, SCPB@DOX NPs, SCPB@DOX@EM NPs or SCPB@DOX@EM@FA NPs (0.5 μg mL−1 DOX) for 4 h. Then, the cells were irradiated with or without an 808 nm NIR laser (0.8 W cm−2) for 5 min and further incubated for 20 h. The treated cells were lysed in the RIPA buffer. After separating with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were transferred into nitrocellulose membranes. The membranes were incubated with antibodies against PARP, IKK, NF-κB, caspase-3, BAX, Bcl-2 and β-actin overnight at 4 °C after blocking with 5% skim milk, followed by incubation with HRP-conjugated secondary antibodies. The bands were visualized using the BIO-RAD ChemiDoc XRS chemiluminescence system.
3
Quantifying Salivary Alpha-Amylase by Western Blot
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sAA amount (μg/ml) was determined by Western blotting. Briefly, saliva samples of equal quantity of total protein were prepared by solubilizing with the SDS-PAGE loading buffer and heating at 100°C for 10 min. For quantification purpose, a human sAA protein sample (Sigma-Aldrich) of known quantity was also run on each gel. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then blocked by 5% milk at room temperature, followed by incubation with rabbit anti-sAA antibody (Abcam) at 4°C overnight. After washing with PBST, the membrane was subjected to incubation with goat anti-rabbit IgG-horseradish peroxidase conjugate (R&D Systems) at room temperature for 1 hour. After washing, the membrane was then exposed to Pierce™ ECL Western Blotting Substrate (Thermo Scientific) for 1 min. A ChemiDoc XRS+ Chemiluminescence system (Bio-Rad) was used for detecting the sAA protein band. The test sAA amount (μg/ml) was estimated by comparing with the respective standard sAA protein of known quantity.
4
Quantification of Apoptotic Proteins
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Cells (1 × 106 cells/well) were cultured in the 6-well plates with EA (10 μM) and C1 (5 μM, 10 μM) treatment for 24 h and harvested. The protein concentration in the total extract was quantitated by the BCA protein assay kit (562 nm). The expression of GST was detected strictly by Western blotting (WB) with different antibodies: β-actin (1:3,000), GST monoclonal antibodies, Bcl-2 monoclonal antibodies, Bax monoclonal antibodies, caspase 3/p17/p19 monoclonal antibodies, and cytochrome C monoclonal antibodies (1:1,000). The detection was carried out on the BIO-RAD ChemiDoc XRS chemiluminescence system (Hercules, CA, USA).
5
PCSK9 Expression and Secretion Analysis
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PCSK9 expression and secretion analysis in HEK293 cells transfected with empty plasmid and the different PCSK9 variants was performed by Western blotting. For that purpose, proteins from cell lysates or the supernatants were resolved by 8.5% Tris-Glycine SDS-PAGE. Gels were next blotted onto Nitrocellulose membranes (Protran BA 83, Whatman™, GE Healthcare, Munich, Germany), blocked for 1 h in TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% BSA and immunoblotted with a rat monoclonal anti-FLAG antibody (1:1000) (Cat. No:MA1–142; Invitrogen, Thermo Fisher Scientific, Pierce, CA, USA) for 16 h at 4 °C. Then, they were counterstained with a horseradish peroxidase-conjugated goat anti-rat antibody (Cat. No: 7077S; Cell Signalling Technology® Inc., Danvers, MA, USA). The signal was created by adding SuperSignal West Dura Extended Substrate (Thermo Fisher Scientific, Pierce, CA, USA) and it was measured by the ChemiDoc XRS chemiluminescence system (Bio-Rad, Hercules, CA, USA) at 425 nm. The relativisation was performed using Glyceraldehide 3-phosphate dehydrogenase (GAPDH) as control.
6
Western Blot Analysis of GSTM3 and GSTP1
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After washing with PBS, harvested cells were lysed in RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche, Switzerland). Protein concentrations were determined with BCA Protein Assay Kit (Solarbio Science & Technology Co., Ltd., Beijing, China). Equal amounts of proteins were subjected to SDS-PAGE and Western blotting. The primary Abs were as follows, anti-GSTM3 Ab (1:500, Proteintech, USA), anti-GSTP1 Ab (1:500, Proteintech, USA) and anti-GAPDH Ab (1:2000, Proteintech, USA). The second Abs were as follows, goat anti-mouse HRP conjugated IgG (1:4000, Proteintech, USA) and goat anti-rabbit HRP conjugated IgG (1:4000, Proteintech, USA). Western blotting signals were detected and developed using a BIO-RAD ChemiDoc XRS chemiluminescence system (Bio-Rad Inc., CA, USA).
7
Protein Expression Analysis in Cells
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Cells or tissues were harvested and lysed by RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with a protease inhibitor cocktail (Solarbio Life Science, China). The concentrations of total protein were detected via a BCA Protein Assay Kit (Solarbio Life Science, China) and equal amounts of protein were loaded into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then proteins were transferred onto Nitrocellulose (NC) membrane (Pall Corporation, USA). Later, NC membranes were incubated with different primary antibodies (anti-α-SMA, anti-CD31, anti-NSE, anti-GAP43, anti-S100β, anti-RhoA and anti-Rock1, ProteinTech Group, Inc) at 4 °C overnight and then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. The signals were detected with the BIO-RAD ChemiDoc XRS chemiluminescence system (Bio-Rad Inc., CA, USA).
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