For protein expression studies, whole cell lysates were extracted using SDS lysis buffer and then supplemented with cOmplete protease inhibitor mixture (Roche Applied Science). Equal amounts of whole cell lysates were separated by SDS-PAGE and immunoblotted with antibodies that recognized HIF-1α (BD Bioscience), OCT4 (BioLegend). Anti-GAPDH (Santa Cruz Biotechnology, Inc.) and anti-Actin (Millipore) antibodies were used to assess equal protein loading. Immunoblots were visualized using an enhanced chemiluminescence detection kit (ECL-Plus, Amersham Pharmacia Biotech) and were imaged with a Versadoc 3000 Imaging System (Bio-Rad). Densitometric data were obtained and quantified with Quantity One Software (Bio-Rad). Western analyses shown are representative of two to four independent experiments.
Versadoc 3000 imaging system
Manufactured by Bio-Rad
Sourced in United States
The VersaDoc 3000 Imaging System is a versatile and compact instrument designed for capturing high-quality images of various samples, including gels and blots. The system utilizes a CCD camera and a selection of optical filters to provide accurate and reproducible imaging results. The VersaDoc 3000 Imaging System is capable of capturing images in various file formats and offers users the ability to adjust exposure time and other imaging parameters to optimize the output.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti actin, by Merck Group (2 mentions)
Ecl plu, by Cytiva (2 mentions)
Complete protease inhibitor mixture, by Roche (2 mentions)
Hif 1α, by BD (2 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Chemilucent ecl detection system, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Roche (1 mentions)
Phospho 4ebp1, by Cell Signaling Technology (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Phospho p70s6k, by Cell Signaling Technology (1 mentions)
Hif 2α, by Novus Biologicals (1 mentions)
Wet tank blotting system, by Bio-Rad (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Protran nitrocellulose transfer membrane, by Cytiva (1 mentions)
Nupage 12 bis tris, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
9 protocols using versadoc 3000 imaging system
1
Protein Expression Analysis by Western Blot
2
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer (CST) containing protease and phosphatase inhibitor cocktails (KeyGEN) on ice. The supernatant was collected after centrifugation, and the concentration was determined by a BCA assay (Thermo Fisher Scientific, USA). A total of 30 μg of protein extract was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk in TBST buffer [10 mM Tris–HCl (pH 7.4) containing 0.1% Tween 20] and was then incubated with a primary antibody at 4 °C overnight (Additional file 1 : Table S3). After washing 3 times, the membrane was incubated with a secondary antibody (CST) at room temperature for 40 min, and immunoreactions were detected with a chemiluminescent substrate (Thermo). Images were acquired with a VersaDoc 3000 Imaging System (Bio-Rad), and densitometric analysis was performed with ImageJ (National Institutes of Health, Bethesda, MD). The relative protein expression was calculated as a ratio of the density of each protein band relative to the loading control GAPDH and was normalized to unstimulated controls.
3
Western Blot Analysis of BAK-1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysis and Western blotting were carried out as previously described [4 (link)]. Primary antibodies against BAK-1 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase-conjugated secondary antibody was obtained from Roche. Protein bands were visualized using the ChemiLucent ECL Detection System (Millipore, Billerica, MA) and were imaged by exposure to x-ray film. Densitometric analysis was performed by using VersaDoc 3000 Imaging system (Bio-Rad) and 1-D analysis Quantity One software (Bio-Rad) to quantify the relative intensities of protein bands.
4
Protein Expression Analysis Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein expression studies, whole cell lysates were extracted by SDS lysis buffer as previously described [20] , [21] (link), and supplemented with Complete protease inhibitor mixture (Roche Applied Science). Equal amounts of whole cell lysates were loaded, the proteins were separated by SDS-PAGE and immunoblotted with antibodies that recognized HIF-1α (BD Bioscience, 610958), HIF-2α (Novus Biologicals, NB100-122), phospho-p70S6K (Thr389, Cell Signaling, 9234), p70S6K (Cell Signaling, 9202), phospho-4EBP1 (Thr37/46, Cell Signaling, 2855), 4EBP1 (Cell Signaling, 9452), hydroxy-HIF1α (Pro564, Cell Signaling, 3434), CAIX (GeneTex, GTX70020). Anti-GAPDH (Santa Cruz Biotechnology, sc-25778) and anti-Actin (Millipore, 2020280) antibodies were used to assess equal protein loading. Immunoblots were visualized by an enhanced chemiluminescence detection kit (ECL-Plus, Amersham Pharmacia Biotech) and were imaged with a Versadoc 3000 Imaging System (Bio-Rad). Densitometric tracing were obtained and quantitated with Quantity One Software (Bio-Rad).
5
Proteomic Analysis of PrP Isoforms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Twenty percent (w/vol) tissue homogenates were prepared in 0.25 M sucrose in PBS using a Ribolyzer (Bio-Rad). Total protein concentration was measured with the BCA Protein Assay (Pierce), according to the manufacturer’s instructions. For the detection of partially PK-resistant PrP, proteins were digested with PK (Roche) at a final concentration of 25 µg/ml. Protein homogenates in NuPAGE LDS sample buffer (Invitrogen) containing β-mercaptoethanol as reducing agent were separated on a NuPAGE 12% Bis-Tris (Invitrogen) using the NuPAGE Gel Electrophoresis System (Invitrogen) and transferred onto a Protran Nitrocellulose Transfer Membrane (Whatman) using the Wet/Tank Blotting System (Bio-Rad), according to the manufacturers’ instructions. Antibodies used were POM1 (200 ng ml–1 [32 (link)]) as primary and HRP-conjugated goat anti-mouse IgG (H+L) (1:17000 dilution, from Invitrogen) as secondary. Blots were developed using HRP substrate (ECL, Pierce) and visualized using the Versadoc 3000 imaging system (Bio-Rad).
6
SDS-PAGE and Western Blot Analysis of Troponin-T
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein concentration of the sample was adjusted to 2 mg/mL using sample
buffer (2× treatment buffer, 2-mercaptoethanol and bromophenole blue, pH
6.8) and heated at 95°C for 5 min. Each sample was loaded into 4%
Stacking Gel and the proteins were separated using 12.5% separating Gel.
20 cm mini gel was run at 100 V. After SDS-PAGE, the protein in the gel
transferred to 0.2 μm PVDF membranes at 200 mA for 60 min in transfer
buffer (10% methanol, 192 mM glycine, 25 mM Tris). Subsequently, the
membrane blocked by 5% skim milk at room temperature for 1 h. The
membrane incubated with monoclonal primary antibodies (Monoclonal
anti-troponin-T (JLT-12, SIGMA, USA), diluted 1:2,500 in TTBS). The bound
antibodies were visualized using ECL kit and then the images were taken by Versa
Doc 3000 imaging system (Bio-Rad, Hercules, USA).
buffer (2× treatment buffer, 2-mercaptoethanol and bromophenole blue, pH
6.8) and heated at 95°C for 5 min. Each sample was loaded into 4%
Stacking Gel and the proteins were separated using 12.5% separating Gel.
20 cm mini gel was run at 100 V. After SDS-PAGE, the protein in the gel
transferred to 0.2 μm PVDF membranes at 200 mA for 60 min in transfer
buffer (10% methanol, 192 mM glycine, 25 mM Tris). Subsequently, the
membrane blocked by 5% skim milk at room temperature for 1 h. The
membrane incubated with monoclonal primary antibodies (Monoclonal
anti-troponin-T (JLT-12, SIGMA, USA), diluted 1:2,500 in TTBS). The bound
antibodies were visualized using ECL kit and then the images were taken by Versa
Doc 3000 imaging system (Bio-Rad, Hercules, USA).
7
Western Blot Analysis of Cytoskeletal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were separated in a 4-10% SDS-PAGE followed by electrophoretic transfer onto PVDF membranes. The membranes were blocked with 5% non-fat milk in TBS-T (137 mM NaCl, 20 mM Tris, 0.1% Tween 20, pH 7.6) and incubated overnight at 4 °C with anti-αSMA (1:500), anti-fibronectin (1:1000), anti-β-actin (1:10000) or antiβ-tubulin (1:10000) antibodies diluted in blocking solution. After three washes with TBS-T, the membranes were incubated for 1 h, at room temperature, with an alkaline phosphatase-linked secondary antibody, specific to rabbit or mouse immunoglobulin G (1:20000, Amersham Biosciences, GE Healthcare, UK). Immunoreactive bands were visualized using ECF substrate in the Versa-Doc 3000 imaging system (Bio-Rad, USA). Densitometry of the bands was quantified using Quantity One Software (Bio-Rad, USA).
8
Brain Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A protein extraction kit (Thermo) was used to obtain brain supernatant. The samples were then separated on SDS polyacrylamide gel and transferred to a PVDF membrane, blocked with BSA for 1.5 h, washed with TBST for 5 min three times, and incubated with different antibodies overnight. The membrane was rinsed three times in TBST for 5 min each time and then incubated with the HRP-labeled antibody for 1 h. The band was detected by ECL chemiluminescence solution, and the protein expression was displayed by Bio-Rad VersaDoc 3000 Imaging System (Bio-Rad, Mississauga, ON, Canada). The original image was obtained in the Supplementary Information .
9
RNA Extraction and RT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA extraction was performed in accordance with the manufacturer's instructions for TRIzol. Synthesis of first-strand cDNA was performed in accordance with the kit. Briefly, l µl oligo (dT) were added to 2 µg total RNA and heated at 70°C for 5 min. A total of 5 µl 5X avian myeloblastosis virus (AMV) buffer, 2.5 µl desoxyribonucleotide triphosphate (10 mmol/l), 1 µl AMV-RT (10 U/µl), l µl RNasin (40 U/µl; Promega Corp.) and diethylpyrocarbonate-treated water were then added to a final volume of 25 µl, mixed and reacted at 42°C for 60 min.
The PCR primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China;Table I ). PCR cycling conditions were as follows: 94°C for 2 min, 35 cycles of 94°C for l min and 58°C for 1 min, followed by extension at 72°C for l min, on a GeneAmp PCR system 9600 (Applied Biosystems). Expression levels were calculated relative to GAPDH levels, which acted as the endogenous control. The PCR products then underwent 1% agarose gel electrophoresis and were observed under an ultraviolet lamp and images were captured on a Bio-Rad VersaDoc 3000 Imaging system (Bio-Rad). Image J software (NIH, Bethesda, MD, USA) was used for grey value analysis.
The PCR primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China;
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!