Western blots were performed as previously described [17 –28 (link)]. Briefly, cells were lysed in RIPA buffer, and protein concentration was determined using the Bradford method. Twenty micrograms of intracellular proteins were loaded and resolved on a 10% SDS–PAGE gel. Antibodies against FoxO3a and NF-kB, atrogin, MuRF-1, myogenin, desmin, and SOD-2 (diluted 1:500 in T-TBS 0.1%, Santa Cruz Biotechnology, Dallas, TX) were used as primary antibodies. Horseradish peroxidase-conjugated donkey anti-mouse and goat anti-rabbit antibodies were used as secondary antibody (diluted 1:5000 in T-TBS 0.1; Santa Cruz Biotechnology, Dallas TX). The signal was detected using the ECL system (Chemicon-Millipore, Milano, Italy). Protein levels were normalized with respect to the expression of the housekeeping protein, actin (diluted 1:1000 in T-TBS 0.1%). The semi-quantitative analysis of protein levels was carried out by the Gel Doc 2000 UV System and the Gel Doc EZ Imager (Quantity one software, Bio-Rad Laboratories).
Gel doc 2000 uv system
Manufactured by Bio-Rad
Sourced in United States, Italy
The Gel Doc 2000 UV System is a compact and versatile imaging system designed for the visualization and documentation of DNA, RNA, and protein gels. The system utilizes a UV transilluminator to excite fluorescent samples, and a high-resolution camera for capturing images of the illuminated gels. The Gel Doc 2000 UV System is a reliable tool for a variety of laboratory applications that require the analysis of nucleic acids or proteins separated by gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc ez imager, by Bio-Rad (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (3 mentions)
Ripa buffer 1, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Polyclonal anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Nf κb, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ecl system, by Merck Group (1 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
6 protocols using gel doc 2000 uv system
1
Western Blot Analysis of Muscle Atrophy Markers
2
Quantitative Analysis of Cytoskeletal Proteins
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Proteins were extracted after 4 h and 24 h using a RIPA lysis buffer and the concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad Laboratories s.r.l.). Equal amounts of protein (40 µg) were loaded onto a polyacrylamide gel for SDS-PAGE and transferred to a nitrocellulose membrane. The filters were incubated overnight at 4 °C with antibodies against elastin (1:250), tubulin (1:500), and actin (1:1000). The membranes were washed three times for 10 min and incubated with anti-rabbit (1:10,000), anti-mouse (1:5000), or anti-goat (1:5000) horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. Protein levels were normalized with respect to the signal of anti-actin polyclonal antibody. Blots were developed using the ECL system (Enhanced Chemiluminescence) (Amersham Biosciences, Little Chalfont Buckinghamshire United Kingdom) according to the manufacturer’s protocol. Densitometric analyses were performed using a Gel Doc 2000 UV System and the Gel Doc EZ Imager (Quantity One software, System ID 4020DABD, Bio-Rad Laboratories s.r.l.).
3
Quantifying Chondrocyte Protein Levels
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Western blot analyses were performed following standard procedures. After 10 days of treatment with BC (1% w/v) and CS (1% w/v), chondrocytes were lysed in RIPA buffer (1×) (Cell Signaling Technology). Protein concentration was determined using the Bradford method [Bradford, 1976 ] and 60 µg intracellular proteins were loaded and resolved using 8% SDS–PAGE. The separated proteins were then transferred to nitrocellulose membrane (Amersham). The membrane was blocked in 5% milk, Tris‐buffered saline and 0.05% Tween‐20. Primary antibodies to detect type II collagen and type I collagen (Abcam) were used at 1:250 dilutions. Immunoreactive bands were detected by chemiluminescence using corresponding horseradish peroxidase‐conjugated secondary antibody (Santacruz Biotechnology; 1:5000 dilutions) and reacted with an ECL system (Chemicon‐Millipore). Protein levels were normalized with respect to the signal obtained with anti actin polyclonal antibody (Santacruz Biotechnology; 1:500 dilutions). The semi‐quantitative analysis of protein levels was carried out by the Gel Doc 2000 UV System and the Gel Doc EZ Imager, using quantity one software (Bio‐Rad Laboratories).
4
Quantitative RT-PCR Analysis of DMT1 Isoforms
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Fifty nanograms of cDNA were selectively amplified for DMT1 +IRE and–IRE transcripts, as previously described [19 (link)], and for the ribosomal protein S18 (RPS18), as housekeeping gene. Amplimers, resolved into 2.0% agarose gel, were detected by “Gel Doc 2000 UV System” (Bio-Rad, Hercules, CA, USA). The quantification of the pixels of all the bands of the amplimers was made by using the quantization software of the same instrument (Quantity One—4.6.5, Bio-Rad, Hercules, CA, USA). The quantification of the +IRE and–IRE bands was performed with respect to the S18 bands, by dividing the signal of the IRE bands for the signal of their relative S18 bands. After normalization with respect to S18, the ratio between +IRE/18S and–IRE/18S values was calculated. These final data were categorized as less or more than one.
5
Western Blot Analysis of COMP-2 and NF-κB Proteins
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The cells’ intracellular total protein content was extracted in the radioimmunoprecipitation assay (RIPA buffer) (1×) (Cell Signaling Technology, USA). Protein concentration was determined using the Bradford method (Biorad Laboratories, Milan, Italy) and 5 μg of proteins was separated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes (Millipore, Bedford, MA), and blocked with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST). The membranes were incubated with primary antibodies to detect COMP-2 (Abcam, Cambridge, UK) and NF-κB (Santa Cruz Biotechnology, CA, USA) both used at 1:500 dilutions and incubated overnight at 4 °C. After washing with TBST, immunolabeling bands were detected by the chemiluminescence detection system using corresponding horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, CAUSA), diluted 1:10,000 for 1 h at room temperature and reacted with an ECL system (Merck Millipore, Darmstadt Germany). Protein levels were normalized with respect to the signal obtained with an anti-actin antibody 1:500 dilution (Santa Cruz Biotechnology, CA, USA). The semi-quantitative analyses of protein levels were carried out using the Gel Doc 2000 UV System (Biorad laboratories, Milan, Italy) according to the manufacturer’s protocols and previously reported [32 , 33 (link)].
6
Protein Expression Analysis Using Western Blot
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Western blot analyses were performed after seven days of treatment with H-HA, L-HA and H/L-HA hybrid complexes. Cells were lysed in RIPA buffer (Cell SignalingTechnology). Protein concentration was determined using the Bradford method [25 (link)] and 80 μg intracellular proteins were loaded and resolved using 10% SDS–PAGE. The separated proteins were then transferred to nitrocellulose membrane (Amersham). The membrane was blocked in 5% milk dissolved in Tris-buffered saline and 0.05% Tween-20. Elastin (60kDa) primary antibody was used at 1:250 dilutions. Immunoreactive bands were detected by chemiluminescence using corresponding horseradish peroxidase-conjugated secondary antibody (Santacruz Biotechnology; 1:5000 dilutions) and reacted withan ECL system (Chemicon-Millipore). Protein levels were normalized respect to the signal of anti actin polyclonal antibody using as housekeeping protein (Santacruz Biotechnology; 1:1000 dilutions). The semi-quantitative analysis of protein levels was carried out by the Gel Doc 2000 UV System and the Gel Doc EZ Imager (Quantity one software, Bio-Rad Laboratories).
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