Western blot analysis was performed using equal amounts of whole cell extract protein as described before33 (link),35 (link). Briefly, cell lysates were run on SDS PAGE, proteins transferred to a nitrocellulose membrane. The membranes were incubated with the primary antibodies, rabbit anti- RGS4 (Santa cruz), mouse anti-SPARC (Haematologic Technologies, Inc), rabbit anti-AKT-S473, rabbit anti-AKT, mouse anti-beta-actin (Cell Signaling), respectively, followed by Horseradish peroxidase labeled donkey anti rabbit or donkey anti mouse antibodies. Protein signal was visualized by using Immun-Star chemiluminescent kit (Bio-Rad) and quantified by Bio-Rad Imager.
Immun star chemiluminescent kit
Manufactured by Bio-Rad
Sourced in United States
The Immun-Star chemiluminescent kit is a lab equipment product designed for the detection and quantification of proteins. It utilizes a chemiluminescent reaction to generate a measurable signal that is proportional to the amount of target protein present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Hyperfilm, by Cytiva (1 mentions)
Protein a agarose beads, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Alkaline phosphatase conjugated secondary antibody, by Merck Group (1 mentions)
Sc 8984, by Santa Cruz Biotechnology (1 mentions)
Nytrann nylon membranes, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
5 protocols using immun star chemiluminescent kit
1
Western Blot Protein Quantification
2
Quantitative Western Blot Analysis
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Western blots were performed using equal amounts of whole cell extract protein. Briefly, cell lysates were run on SDS PAGE, proteins transferred to a nitrocellulose membrane. The membranes were incubated with the primary antibodies anti- TXNIP, anti-phospho-Ets1and anti-GAPDH, respectively, followed by Horseradish peroxidase labeled donkey anti rabbit or donkey anti mouse antibodies. Protein signal was visualized by using Immun-Star chemiluminescent kit (Bio-Rad) and quantified by Bio-Rad Imager.
3
Protein Carbonyl, uNOS, and Nitrotyrosine Detection
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Proteins were separated by 10% SDS–PAGE as reported [37 (link)]. Gels were transferred to PVDF membranes for protein carbonyl and uNOS immunodetection or nitrocellulose membrane for nitrotyrosine immunodetection. Proteins were electroblotted by a semi-dry Trans-Blot cell. Membranes were incubated with a polyclonal antibody against DNP diluted 1:5000, uNOS diluted 1:1000 and nitrotyrosine diluted 1:1000. For PVDF membranes, immunoreactive bands were detected using a photographic film (Hyperfilm, Amersham Pharmacia Biotech) with an enhanced chemiluminescence kit (Immun-Star ChemiluminescentKit, Bio-Rad, Hercules, CA, USA) and for nitrocellulose membrane, the LiCOR Odyssey system was used for immunodetection.
4
Immunoblotting and Immunoprecipitation of IRS2 and SH2B1
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Lysed islets in lysis buffer containing a protease inhibitor cocktail (Roche Diagnostics) were boiled for 5 min and then loaded and electrophoresed on 10–12% SDS-PAGE. Antibodies used for immunoblotting are listed in Supplementary Table 1 . Protein-band densitometry was determined using National Institutes of Health ImageJ software version 1.49d (33 (link)) (freely available at https://imagej.nih.gov/ij/ ) and normalized to tubulin/actin in the same membrane. Immunoprecipitation experiments were carried out using 300 μg of total protein for each sample, with 2 μg of IRS2 antibody and 50 μL of protein A agarose beads (Sigma-Aldrich). All immunoblotting images were developed using an Immun-Star Chemiluminescent Kit (Bio-Rad Laboratories). For pulse experiments, cells were pulse-labeled with [35S]-methionine for 2 h followed by immunoprecipitation with IRS2 or SH2B1 antibodies.
5
Western Blot Analysis of SREBP1
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Fifty microgram of protein extract from liver samples was loaded per lane and submitted to electrophoresis in a 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel. When electrophoresis was completed, the gel was equilibrated in transfer buffer (25 mM Tris-HCl, 192 mM glycine, 20 % methanol, pH 8.3), and electroelution proceeded at 4 °C onto NytranN nylon membranes (Whatman, Kent, UK) for 2 h at 60 V. A rabbit polyclonal antibody against rodent SREBP1 was used as the primary antibody (sc-8984, Santa Cruz Biotechnology, Dallas, TX, USA; 1:500). The Immun-Star Chemiluminescent Kit (Bio-Rad, Hercules, CA, USA) was used to immunodetect SREBP1 with an alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich, Saint Louis, MO, USA; 1:3000).
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