Protein concentration was determined by PIERCE BCA protein assay (Fisher Scientific). Samples were heated for 10 min at 95°C in sample buffer (2.0ml 1M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% 14.7 M β-mercaptoethanol 12.5mM EDTA, 0.02% bromophenol blue). Proteins were then separated on 12% Bis-acrylamid mini gels containing 0.1% 2,2,2-Trichloroethanol for subsequent visualization of protein loading using a Chemidoc MP imaging system (Biorad). Following protein transfer onto PVDF membranes (Biorad), transfer efficiency was checked using a Chemidoc MP imaging system (Biorad). The membranes were blocked for 60 min in 1% bovine serum albumin (in phosphate-buffered saline containing 1% Tween 20 (PBST); 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.76mM K2HPO4; pH 7.4) and sequentially incubated with the primary and secondary antibodies. A 1:1000 dilution for the primary antibody (rabbit anti-AQP4, Chemicon, as used for immunostainings; mouse anti-β-tubulin, Sigma Aldrich), and 1:10,000 for the HRP-labeled secondary antibody (anti-rabbit and anti-mouse IgG, Cell Signaling) were utilized. All antibodies were diluted in 1% bovine serum albumin in PBST.
Chemidoc mp imaging system
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, China, Germany, Canada, Australia, France, Italy, Austria, Japan, Spain, Denmark, Portugal, Belgium, Sweden, United Arab Emirates, Israel
The ChemiDoc MP Imaging System is a versatile laboratory instrument designed for the detection and analysis of various biomolecules, including proteins, nucleic acids, and chemiluminescent samples. It utilizes advanced imaging technology to capture high-quality images and data for applications such as Western blotting, gel documentation, and DNA/RNA visualization.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (316 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (204 mentions)
Pvdf membrane, by Merck Group (167 mentions)
Clarity western ecl substrate, by Bio-Rad (160 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (156 mentions)
Trans blot turbo transfer system, by Bio-Rad (130 mentions)
Ripa buffer, by Thermo Fisher Scientific (113 mentions)
Nitrocellulose membrane, by Bio-Rad (103 mentions)
Pvdf membrane, by Bio-Rad (93 mentions)
Image lab 5, by Bio-Rad (63 mentions)
Image lab 6, by Bio-Rad (58 mentions)
Protease inhibitor cocktail, by Merck Group (57 mentions)
Laemmli sample buffer, by Bio-Rad (54 mentions)
Gapdh, by Cell Signaling Technology (49 mentions)
Image lab, by Bio-Rad (48 mentions)
β actin, by Merck Group (47 mentions)
Ripa lysis buffer, by Beyotime (45 mentions)
Trans blot turbo system, by Bio-Rad (45 mentions)
Protease inhibitor cocktail, by Roche (45 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (44 mentions)
3 639 protocols using chemidoc mp imaging system
1
Protein Quantification and Western Blotting
2
SDS-PAGE Analysis of Fermentation Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE of fermentation supernatant samples withdrawn at different time points was performed according to standard conditions (Laemmli, 1970 (link)). After staining with Coomassie Brilliant Blue R-250, SDS PAGE gels were imaged using a ChemiDoc (Chemidoc™ MP Imaging System, Bio-Rad). Stain free SDS-PAGE gels were directly visualized using a ChemiDoc (Chemidoc™ MP Imaging System, Bio-Rad) without further treatment. Deglycosylation of Thc_Cut1 and Thc_Cut1_ko mutants was performed using Endo Hf according to manufacturer's instructions (New England Biolabs). Glycostain gels were prepared according to Pro-Q® Emerald 300 Glycoprotein Gel and Blot Stain Kit manual and detected by G-Box or hand-held UV lamp at 300 nm.
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen livers and quadriceps muscles were crushed using a mortar and pestle under liquid nitrogen and homogenized in 1 ml ice-cold 1% NP40, 1 mM EDTA in phosphate buffered saline (PBS), supplemented with complete EDTA free protease inhibitor tablets and PhosSTOP phosphatase inhibitor tablets (Roche), and centrifuged at 10,000 g for 10 min. The protein concentrations of supernatants were quantitated using the DC protein assay (Bio-Rad). Samples were resolved on 4–15% SDS-PAGE TGX Stain-Free gels (Bio-Rad) and transferred onto nitrocellulose membrane using a Trans Turbo Blot system (Bio-Rad), after imaging for total protein using the Stain-Free imaging program on the ChemiDoc MP Imaging System (Bio-Rad). Immuno-blotting was performed with rabbit polyclonal antibodies to neutrophil elastase (ab68672, Abcam), cysteine dioxygenase type 1 (ab53436, Abcam) and cysteine sulfinate decarboxylase (ab101847, Abcam) all dissolved 1:1000 in 5% bovine serum albumin (BSA). HRP-conjugated goat anti-rabbit secondary antibodies were from Thermo Fisher Scientific. Chemiluminescence signal was captured using the ChemiDoc MP Imaging System (Bio-Rad). Resultant images were quantified using ImageJ software [31 (link)]. A common sample was loaded onto each gel to normalise for detection efficiencies across membranes, and all bands were standardised to total protein for that lane [32 (link)].
4
Multichannel Blot Imaging and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The blots were first imaged using the multichannel feature of the ChemiDoc MP Imaging System (Bio-Rad) with “Blots”, “Alexa 647”, “Alexa 488” and “Colorimetric” applications using the auto expose settings. This was followed by chemiluminescence detection using the Clarity or Clarity Max Western ECL Substrate (Bio-Rad – 1705062) and the ChemiDoc MP Imaging System with “Blots”, “Chemiluminescence” application and auto expose or manual settings. All densitometric data were acquired and translated using Image Lab software (Bio-Rad) with data analysis, graphing and statistics using GraphPad Prism software.
5
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in reducing Laemmli SDS sample buffer containing PhosSTOP (Phosphatase Inhibitor Cocktail Tablets, Roche, Switzerland) at 96°C for 10 min and the proteins separated on NuPAGE® Novex® 4–12% Bis–Tris Gels. Subsequently, the proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA), the membranes were quenched and proteins were detected using specific antibodies. Labelled protein bands were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, USA) and ChemiDoc MP Imaging System (Bio‐Rad) CCD camera. Protein band intensities were quantified using ChemiDoc MP Imaging System and Image Lab software (Bio‐Rad, Hercules, CA, USA).
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins (40 µg) were separated on a 7.5% stain-free SDS-polyacrylamide gel by electrophoresis then transferred to mini low fluorescence PVDF membranes (Bio-Rad Laboratories, Hemel Hempstead, UK) and visualised using the Chemidoc MP imaging system (Bio-Rad Laboratories). The membrane was blocked with 3.5% (w/v) TBS-Tween (10 mmol/L Tris pH 8, 150 mmol/L NaCl, 0.1% Tween 2.0) for 1 h at room temperature (RT). Appropriate primary antibody was added and incubated overnight at 4°C (anti-eNOS 1:200, anti-peNOSser1177 1:200, anti-O-GlcNAc 1:200; Santa Cruz Biotechnology, Dallas, TX, USA; anti-AMPKα 1:1,000 and anti-pAMPKα 1:1,000; Cell Signalling, Hitchin, UK) followed by exposure to a horseradish peroxidase-conjugated secondary antibody for 1 h at RT. Chemiluminescence was performed with Clarity Western ECL Substrate (Bio-Rad Laboratories) and images were obtained using the Chemidoc MP imaging system. Immunoblots were analysed using ImageLab 5.2.1 (Bio-Rad Laboratories) software and adjusted to the total protein of the sample as previously described [35 (link)].
7
Insulin-mediated signaling in myotubes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The myotubes were incubated for 20 min in DMEM-low glucose-Glutamax in the presence or absence of 100 nM insulin. Afterwards, the cells were harvested in a RIPA buffer (Sigma, St. Louis, MO, USA) complemented with 10 µL/mL protease inhibitor, 10 µL/mL phosphatase I inhibitor and 10 µL/mL phosphatase II inhibitor. Afterwards, the protein concentration was determined by BCA reagent (Thermo Scientific, Rockfold, IL, USA). 20 µg of total protein were run on 4–15% Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad, Hercules, CA, USA), electroblotted onto PVDF membranes (Millipore, Bradford, MA, USA), and immunodetected with ChemiDoc MP imaging system (BioRad, Hercules, CA, USA) while using the following primary antibodies: GLUT4 (Santa Cruz Biotech, CA, USA), Ser9 pGSK-3β, Thr172pAMPK, Ser473 pAkt (Cell Signaling Technology, Danvers, MA, USA), Tyr-989 pIRS-1 (Abcam, Cambridge, UK), and Thr642 pAS160 (Gene Tex, CA, USA). Histone H3 (Cell Signalling Technology, Danvers, MA, USA) served as an internal control, with the exception of pAkt, where the internal control was GAPDH (Cell Signalling Technology, Danvers, MA, USA). The bound antibodies were visualized by an ECL system (Thermo Fisher Scientific Inc., Rockford, IL, USA) and quantified using Chemi-Doc MP imaging system (Bio-Rad, Hercules, CA, USA).
8
Stain-Free Protein Visualization and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were separated on SDS-PAGE supplemented with 0.5% of 2,2,2-Trichloroethanol to allow the stain-free visualization of proteins migrated at 40 mA/gel. Using the stain-free option in the Image Lab software (RRID: SCR_014210) of the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA, RRID: SCR_019037) enabled the visualization of proteins in gel or on the 0.45 µm PVDF membrane after protein transfer using Trans-blot Turbo (Bio-Rad, Mississauga, ON, Canada). The membrane was blocked with 5% skim milk dissolved in TBS-T buffer (20 mM Tris–HCl, 140 mM NaCl, 0.3% Tween-20) before incubation with primary antibody diluted in TBS-T with 0.5% NaN3 for more than 1 h, followed by 1 h incubation with HRP-conjugated secondary antibody diluted in TBS-T. Immunoblotted proteins were visualized using ECL substrate (Bio-Rad) with ChemiDoc MP Imaging System (Bio-Rad) using chemi function in Image Lab software version 6.0.1 (Bio-Rad).
9
Western Blot Analysis of Phosphoproteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phosphorylated proteins were separated using SDS-PAGE (7.5–15% separating gel), transferred to membranes (1620177, BioRad) using a TransBlot Turbo Transfer System (1704150, BioRad), and blocked in 5% BSA (in TBST, A2153-1KG, Sigma-Aldrich). Blots were incubated overnight in rabbit anti-mouse primary antibody for phosphorylated protein of interest (Supplemental Table S5 ), then incubated in a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ab6721, Abcam). Phosphorylated proteins of interest were detected using Clarity Western ECL Blotting Substrate (1705061; BioRad). Images were captured using a ChemiDoc MP Imaging System (1708280; BioRad). Blots were incubated in stripping buffer (21059, ThermoFisher Scientific), washed twice in TBST, then blocked in 5% BSA (in TBST, pH 7.4, A2153; Sigma-Aldrich). Blots were probed with the rabbit anti-mouse primary antibody binding to the total protein (phosphorylated and unphosphorylated; Supplemental Table S5 ) protein of interest. Total protein was detected by incubating blots in Clarity Western ECL Blotting Substrate (1705061; BioRad). Images were captured using a ChemiDoc MP Imaging System (1708280; BioRad).
10
Western Blot Protein Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal concentrations of TBS fraction of the protein extracts (15 μg) were heated with sample buffer (3.2% SDS, 32% glycerol, 0.16% bromophenol blue, 100 mM Tris–HCl, pH 6.8, 8% 2-mercaptoethanol w/v) and separated on Bio-Rad Criterion Stain-free 4–20% SDS-PAGE gels. The gels were activated for 1 min using Bio-Rad chemiDoc MP imaging system prior to transfer of proteins to a 0.45-μm PVDF membrane. The membranes were imaged for total protein using Bio-Rad chemiDoc MP imaging system. Subsequently, the membranes were blocked with 5% milk powder in TBST for 1 h at room temperature and incubated overnight in apoD primary antibody (Santacruz, sc-373965, 1:2000) prior to protein detection using horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) with enhanced chemiluminescence (Amersham ECL Plus Western Blot Detection System, GE Healthcare). The protein band in each gel lane was normalized to total protein using Bio-Rad image lab software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!