After 2D PAGE electrophoresis the gels were placed in a ChemiDoc Touch Imaging System (Bio-Rad) and activated for 45 s in order to enable stain-free visualization of the total proteins. The proteins were then electrotransferred from the gels onto a 0.45 µm nitrocellulose membranes (60 V for 2 h in a Bio-Rad Mini Protean II trans-blot cell operating in a cold room) and the stain-free quantitative images of the transferred proteins were captured in the ChemiDoc Touch Imaging System (Bio-Rad). Membranes were then blocked for 1 h in a 2% skim milk dissolved in a tris-buffered saline supplemented with 0.05% of Tween-20 (TBS-T), and incubated for 2 h in 1:2 000 anti-phosphotyrosine primary antibody (4G10 Platinum, Millipore). After extensive washing in TBS-T, the membranes were incubated for 1 h in 1:30 000 anti-mouse HRP Conjugate secondary antibodies (Bio-Rad). Following the extensive washing in TBS-T the membranes were developed with the Clarity Western ECL Substrate (Bio-Rad) and the chemiluminescence of the phosphotyrosines was captured in ChemiDoc Touch Imaging System (Bio-Rad). The obtained images were analyzed in the Image Lab 5.0 software (Bio-Rad) and the stain-free images of the total proteins transferred to the membranes were used to normalize the signal from the separate samples.
Chemidoc touch imaging system
Manufactured by Bio-Rad
Sourced in United States, China, Germany, United Kingdom, Italy, Japan, Singapore, Canada, Australia, Netherlands, France
The ChemiDoc Touch Imaging System is a laboratory imaging device designed to capture and analyze images of gels, blots, and other samples. It features a touch screen interface and supports a range of imaging techniques, including chemiluminescence, fluorescence, and colorimetric detection.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (171 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (124 mentions)
Pvdf membrane, by Merck Group (118 mentions)
Clarity western ecl substrate, by Bio-Rad (110 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (103 mentions)
Pvdf membrane, by Bio-Rad (64 mentions)
Trans blot turbo transfer system, by Bio-Rad (55 mentions)
Nitrocellulose membrane, by Bio-Rad (47 mentions)
Ripa buffer, by Thermo Fisher Scientific (46 mentions)
Protease inhibitor cocktail, by Merck Group (45 mentions)
Protease inhibitor cocktail, by Roche (41 mentions)
Ripa lysis buffer, by Beyotime (37 mentions)
Bca protein assay kit, by Beyotime (35 mentions)
Image lab 5, by Bio-Rad (34 mentions)
Image lab, by Bio-Rad (32 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (30 mentions)
β actin, by Santa Cruz Biotechnology (29 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (29 mentions)
Polyvinylidene fluoride membrane, by Merck Group (28 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (27 mentions)
1 850 protocols using chemidoc touch imaging system
1
Quantitative Phosphotyrosine Profiling
2
m6A-specific RNA Immunoblotting
3
Immunoblotting and Lectin Blotting Protocols
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For immunoblotting, proteins were separated by SDS-PAGE (Genscript) and transferred onto nitrocellulose membranes (Amersham). Membranes were blocked with 3% bovine serum albumin (BSA) and then probed with speci c primary antibodies and then with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch). The bands were visualized with chemiluminescence (Clarity Western ECL Substrate, Bio-Rad) and imaged by a ChemiDoc Touch imaging system (Bio-Rad). Representative blots are shown from several experiments.
For detection of glycoprotein bands on blots using WGA-HRP. The blots were blocked with 3% bovine serum albumin (BSA) for 1h and were probed with WGA-HRP in PBS-T for 1h at room temperature. The blots were washed 5-times for 10min each with PBS. After ve washes, the blots were visualized with chemiluminescence (Clarity Western ECL Substrate, Bio-Rad) and imaged by a ChemiDoc Touch imaging system (Bio-Rad).
For detection of glycoprotein bands on blots using WGA-HRP. The blots were blocked with 3% bovine serum albumin (BSA) for 1h and were probed with WGA-HRP in PBS-T for 1h at room temperature. The blots were washed 5-times for 10min each with PBS. After ve washes, the blots were visualized with chemiluminescence (Clarity Western ECL Substrate, Bio-Rad) and imaged by a ChemiDoc Touch imaging system (Bio-Rad).
4
SDS-PAGE Protein Separation and Quantification
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Protein concentration was measured by Pierce 660nm protein assay (Pierce). A total of 20 μg surface proteins in a total volume of 10 μL of PBS was mixed with an equal volume (10 μL) of 2x Laemmli buffer with 5% β-mercaptoethanol (Bio-Rad Laboratories, Hercules, CA). The mixture was heated at 95 °C for 5 min. The protein samples were run in a 12% pre-casted stain-free protein gel or 12% pre-casted regular protein gel (Bio-Rad) (Laemmli, 1970 (link); Schagger and von Jagow, 1987 (link)) in a mini protein electrophoresis tank (Bio-Rad) at 110 V with a constant current for 90 min. Protein bands in stain-free gel were scanned with ChemiDoc® Touch Imaging System (Bio-Rad). Protein band images in the SDS-PAGE were captured using a ChemiDoc™ Touch Imaging System (Bio-Rad) and analyzed using Image Lab Software version 5.2.1 (Bio-Rad).
5
Corneal STRA6 Protein Expression Analysis
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Corneal epithelial cells from B6 and vitamin A deficient mouse were scrapped and placed in cell lysis buffer (Part no. 895347, R&D Minneapolis, MN, USA). Protein concentration was determined using a micro bicinchronic acid (BCA) protein assay (Cat# 23235, Thermo Fisher, Waltham, MA, USA). Here, 50 µg of corneal extract was resuspended in sodium-dodecyl sulphate (SDS) sample buffer, boiled for 5 min, and analyzed on 4–15% mini-protean TGX™ stain-free gels (Cat# 4568084, Bio-Rad, Hercules, CA, USA). The proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Cat# 170-4157). The blots were incubated with an anti-STRA6 (Cat# 22001-1-AP, 1:100 dilution, Proteintech Group, Rosemont, IL, USA) or an anti-β actin antibody (Sigma Aldrich, St. Louis, MO, USA, Cat# A5441) overnight. After secondary antibody incubation, the signals from antigen–antibodies complexes were developed with ECL plus Western Blotting Detection kit (Cat# RPN2106, ECL, GE Healthcare, Chicago, IL, USA). Images were taken using ChemiDoc Touch Imaging System (ChemiDoc Touch Imaging System; Bio-Rad, Hercules, CA, USA), and band densities were measured by Bio-rad software (Image lab, v. 6.0; Bio-Rad).
6
Quantitative Western Blot Analysis
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Cells were lysed in 2X Laemmli buffer and stored at -20°C until use. The DC Protein Assay (Bio-Rad) was used to quantify protein concentration as per the manufacturer’s instructions. 10–15 μg of protein lysate was resolved by SDS-PAGE on TGX Stain-Free acrylamide gels (BioRad). Total protein images were acquired from the PVDF membranes after transfer on the ChemiDoc Touch Imaging system (BioRad). Membranes were blocked in 5% BSA in TBS-T (Tris-buffered saline 0.1% Tween-20). Primary and secondary antibodies were diluted in 2.5% BSA, dilutions can be found in Table 2 . Membranes were visualized using Clarity Western ECL substrate and the ChemiDoc Touch Imaging system (BioRad). Densitometry was conducted using ImageJ, area under the curve for each band was calculated, normalized to the respective beta actin loading control band, and presented as fold change.
7
Formulation and Characterization of GP-H3 Vaccine Nanoparticles
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GP-based vaccine formulations were prepared by a simple mixing/adsorption approach. The loading capability of H3 on the GP nanoparticles was evaluated via reducing SDS-PAGE. We prepared a series of GP-H3 nanoparticles at six different weight ratios (2:1, 1:1, 1:2, 1:4, 1:6, and 1:8) under stirring for 20 min at room temperature. The GP-H3 nanoparticles were collected by centrifugation at 15,000 rpm for 20 min and then dispersed in an equal amount of phosphate-buffered saline (PBS). The supernatants were kept separately. Both the nanoparticles and supernatants for all the formulations were analyzed by 10% SDS-PAGE. The gel was stained with Coomassie blue and imaged with the ChemiDoc Touch imaging system (Bio-Rad).
In some formulations, CpG was coloaded onto GP nanoparticles with antigens. We employed agarose gel electrophoresis to investigate whether all the feeding CpG were complexed and loaded onto the GP nanoparticles. Soluble CpG, GP-H3/CpG (10:5:1), GP-H3/CpG (10:5:2.5), and H3+CpG mix were centrifuged at 15,000 rpm for 20 min, and the supernatants were analyzed by 1% agarose gel electrophoresis. The gel was visualized with ChemiDoc Touch imaging system (Bio-Rad) to determine the existence of CpG in the supernatants.
In some formulations, CpG was coloaded onto GP nanoparticles with antigens. We employed agarose gel electrophoresis to investigate whether all the feeding CpG were complexed and loaded onto the GP nanoparticles. Soluble CpG, GP-H3/CpG (10:5:1), GP-H3/CpG (10:5:2.5), and H3+CpG mix were centrifuged at 15,000 rpm for 20 min, and the supernatants were analyzed by 1% agarose gel electrophoresis. The gel was visualized with ChemiDoc Touch imaging system (Bio-Rad) to determine the existence of CpG in the supernatants.
8
Mitochondrial Protein Immunoblotting Protocol
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Mitochondrial suspensions or MDV fractions were collected in SDS‐PAGE buffer (6X Laemmli buffer), vortexed, boiled at 95°C for 5 minutes, and stored at −80°C. Equal volumes of samples were loaded onto a stain‐free gel (TGX Stain‐Free FastCast, 10%, 1.5 mm thick) and electrophoresis was performed at 150 V for 1 hour. PVDF membranes were activated in 100% methanol for 20 seconds, transferred to water for 2 minutes, and left in cold transfer buffer until use. Gels were activated using UV light for 45 seconds in a ChemiDoc Touch imaging system (Bio‐Rad) at high power and 365 nm wavelength. Proteins were transferred onto the membrane using a Trans‐Blot Turbo transfer system (Bio‐Rad) at 1.3 A and up to 25 V for 10 min. Stain‐free images were taken by exposing membranes to UV light using the above‐mentioned settings. Membranes were probed with an anti‐PDHE2/E3bp antibody (ab110333, Abcam) using Snap ID 2.0. Bands were visualized via chemiluminescence using Clarity Western ECL substrate (Bio‐Rad) and imaged under a ChemiDoc Touch imaging system (Bio‐Rad).
9
Western Blot Analysis of HSV-1 Protein Expression
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The measurement of protein expression was done by western blot analysis as described previously [21 (link)]. Vero and SK-N-SH cells (1 × 105 cells/well) infected with HSV-1 strains (MOI = 0.01) were harvested in RIPA buffer, and equivalent protein samples were separated on 8–15% SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated with primary antibodies at 4 °C overnight, followed by HRP-conjugated secondary antibodies at room temperature for 1 h. After washing with TBS-T, protein expression was detected by enhanced chemiluminescence (ECL) using the ChemiDoc™ touch imaging system. Protein expression was normalized to β-actin and quantified using ImageJ software. Data are representative of at least three independent experiments.
10
Western Blot Analysis of Ischemic Myocardium
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The extracted protein lysates from ischemic myocardial tissues and cardiomyocytes were used for Western blot analysis as described previously [27 (link)]. The lysates were normalized to equal amounts of protein, and 10 μg protein from cell lysates or 30 μg protein from tissue lysates were separated by 10% or 12% SDS–PAGE, transferred to polyvinylidene difluoride membrane, and then sealed with 5% nonfat milk in TBST for 3 h. Membranes were incubated with the following primary antibodies: p-AMPK (1:2000, #ab133448, Abcam), AMPK (1:1000, #ab80039, Abcam), PGC-1α (1:1000, #ab191838, Abcam), SIRT3 (1:1000, #ab264041, Abcam), SOD2 (1:5000, #ab13533, Abcam), Bax (1:2000, #ab182733, Abcam), B-cell-lymphoma protein 2 (Bcl-2) (1:2000, #ab182858, Abcam), caspase3 (1:2000, #ab184787, Abcam), cleaved-caspase3 (1:5000, #ab214430, Abcam), and β-tubulin (1:20,000, #66240-1-lg, Proteintech) at 4 °C overnight. Proteins were then probed with specific HRP-conjugated secondary antibodies for 30 min at room temperature. Finally, the protein bands were obtained using the ChemiDoc Touch Imaging System (Chemi Doc, Bio-Rad, Hercules, CA, USA), and the relative intensity of the bands was quantified using Image Lab software.
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