Mitochondrial proteins were extracted according to published protocols [65 (link)] with a few modifications. The extraction buffer was supplemented with 0.02% N-lauroylsarcosine and 0.02% Triton X-100 [66 (link)]. 0.75, 1.5, 3 and 6 µg of mitochondrial proteins were separated by electrophoresis on 15% SDS-containing polyacrylamide gels. The gels were either stained with Coomassie Brilliant Blue (Serva, Heidelberg, Germany) or blotted onto polyvinylidene difluoride membranes (Hybond-P, GE Healthcare, Buckinghamshire, UK). For blotting experiments, the separated proteins were transferred onto polyvinylidene difluoride membranes (Hybond-P; GE Healthcare, Buckinghamshire, UK) using the tank blot system (Perfect Blue Web M, PeqLab, Erlangen, Germany) and a standard transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3). Membranes were treated with blocking buffer (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20, and 0.5% BSA) overnight and subsequently incubated for 1 h with COX2 monoclonal antibody, diluted in buffer (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20). COX2 antibody dilution was 1:1000. Secondary rabbit anti-mouse IgG conjugated to horseradish peroxidase were detected with the ECL Plus proteins gel blotting detection system (GE Healthcare, Buckinghamshire, UK).
Coomassie brilliant blue
Manufactured by Serva Electrophoresis
Sourced in Germany
Coomassie Brilliant Blue is a dye used in protein electrophoresis. It is a blue dye that binds to proteins, allowing their visualization and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Hybond p, by GE Healthcare (1 mentions)
Perfect blue web m, by Avantor (1 mentions)
Hybond, by Cytiva (1 mentions)
Nupage novex bis tris 4 12 pre cast gels, by Thermo Fisher Scientific (1 mentions)
E coli bl21 gold de3, by Agilent Technologies (1 mentions)
Lambda bio, by PerkinElmer (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
4 protocols using coomassie brilliant blue
1
Extraction and Immunoblotting of Mitochondrial Proteins
2
Recombinant Protein Expression in E. coli
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Example 3
TriA and variants thereof were produced in E. coli BL21(DE3) Gold (Agilent Technologies, Germany). Therefore E. coli was transformed with appropriate pET24d N-HIS tag expression vector and chaperone plasmid pGro7 (chaperones groEL and groES). Bacterial strains were grown at 30° C. in 100 mL LB for 20 h and protein expression induced with 0.1 mM IPTG at 25° C. for 20 hs. Cells were harvested by centrifugation at 3000 rpm at 4° C. for 20 min, resuspended in Bug Buster protein extraction reagent (Novagen, Germany) according to manufactures instructions. Lysates were clarified by centrifugation. Samples of bovine serum albumin (5, 10, and 20 g) were loaded onto each gel analyzed by densitometry to provide an internal standard. Protein determinations were verified using Coomassie protein assay dye, according to manufactures instruction (Thermo Scientific; USA). The HIS-tagged enzymes were purified by metal ion affinity chromatography using Ni-IDA 1000 kit (Macherey-Nagel, Germany) following manufactures instructions. Protein purity was accessed by SDS-PAGE using NuPAGE Novex 4-12% Bis-Tris pre-cast gels (Life Technologies; USA) stained with Coomassie Brilliant Blue (Serva, Germany). Protein concentrations were estimated by measuring absorbance at 280 nm using Lambda Bio+ (Perkin Elmer, USA).
3
Analytical Gel Filtration of sNASP Complexes
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Analytical gel filtration of sNASP complexes with monobodies was carried out under 200 mM NaCl, 20 mM HEPES–KOH pH 7.5 and 1 mM EDTA on a Superdex 200 10/300 GL column (GE Healthcare). 30 uM of each component was made up in the same buffer with 2 mM DTT, with monobodies being added first followed by sNASP, histones and then ASF1A. Peaks from the elution profiles were separated out on SDS-PAGE gel and stained with Coomassie Brilliant Blue (SERVA).
4
Bacterial Membrane Protein Isolation
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The bacteria were harvested in the late exponential growth phase (OD600 of 1.0) by centrifugation for 20 min at 6,500 × g and 25 °C. The bacterial pellet was re-suspended in 50 ml of 10 mM HEPES, pH 7.4, containing protease inhibitor cocktail (4 mini-tablets of Complete, EDTA-free, Roche) and DNase I/RNase A (20 μg/ml each). Bacteria were disrupted by four passages through a high-pressure cell disruption system (Model TS, 0.75 KW, Constant Systems Ltd.) at 40,000 psi. The cellular debris was removed by centrifugation at 8,000 × g for 30 min at 4 °C, and the membranes were sedimented from the cleared lysate at 150,000 × g for 2 h at 4 °C. The supernatant (cytosolic fraction) was stored, and the total membrane fraction was washed three times with 10 mM HEPES, pH 7.4. The membrane pellet was subsequently re-suspended in 10 mM HEPES, pH 7.4. The protein concentrations of all samples, i.e. cleared lysate, cytosolic fraction and total membranes, were determined using Bio-Rad’s protein assay reagent. The purity of the fractions was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) using a 10% gel following staining with coomassie brilliant blue (SERVA Electrophoresis GmbH, Heidelberg, Germany).
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