The proteins in each fraction were visualized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 4% to 12% polyacrylamide premade gradient gels (Novex NuPAGE; Invitrogen, Grand Island, NY) with 3-(N-morpholino)propanesulfonic acid as the running buffer (Invitrogen) followed by staining with Coomassie. EZ-Run Rec Protein Ladder (Fisher Scientific) was used as a protein standard. Coomassie Protein Assay Reagent (Thermo Scientific, Waltham, MA) was used to determine protein concentrations with bovine serum albumin as a standard.
Coomassie protein assay reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Coomassie Protein Assay Reagent is a colorimetric assay used to quantify protein concentrations in aqueous solutions. The reagent contains Coomassie Brilliant Blue G-250 dye, which binds to proteins in an acidic environment, resulting in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Ripa buffer, by Merck Group (7 mentions)
Nupage novex, by Thermo Fisher Scientific (6 mentions)
Enhanced chemiluminescence reagent, by PerkinElmer (6 mentions)
Ez run rec protein ladder, by Thermo Fisher Scientific (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by GE Healthcare (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Exobacteria omv isolation kit, by System Biosciences (3 mentions)
Microplate reader, by BMG Labtech (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Biospectrum 810 imaging system, by Analytik Jena (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Ripa lysis buffer, by Cell Signaling Technology (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Mops running buffer, by Thermo Fisher Scientific (2 mentions)
Biospectrum imaging system, by Analytik Jena (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Trisodium citrate dihydrate, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
83 protocols using coomassie protein assay reagent
1
Protein Visualization and Quantification
2
Western Blot Protein Analysis Protocol
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Cells lysates were prepared in ice-cold RIPA buffer (Sigma-Aldrich) with freshly added protease inhibitor cocktail. The lysate was then centrifuged at 12000 g for 10 min at 4°C and the supernatant (total cell lysate) was collected, aliquoted and stored at −80°C. The protein concentration was determined using Coomassie Protein Assay Reagent (Thermo, Rockford, IL). About 40 μg cellular proteins were separated using 6%–12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). Membranes were blocked with 5% fat-free dry milk in 1X Tris-buffered saline (TBS) and incubated with antibodies. Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (Kirkegaard and Perry Laboratories, Gaithersburg, MD) and visualized with enhanced chemiluminescence reagent (Perkin Elmer, Boston, MA).
3
Protein Extraction and Western Blot Analysis
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Cells were washed with ice-cold PBS and homogenized at 4 °C in 20 mM HEPES (pH 7.4) buffer containing 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Igepal, 20% glycerol, and protease and phosphatase inhibitor cocktails. Protein concentration was estimated by Coomassie Protein assay reagent (Thermo Fisher Scientific), with reference to bovine serum albumin (BSA) standards. Total protein extracts (50 µg/lane) were resolved by 7.5% or 10% SDS–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon P, Millipore, Bedford, MA, USA). Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for pTyr705STAT3, Cleaved Caspase-3, PARP, β-actin (Cell Signalling Technology, Beverly, MA, USA), STAT3, Cyclin D1, Survivin, TWIST1 (Santa Cruz, Santa Cruz Biotechnology, Dallas, TX, USA), XIAP, Vimentin and ZEB1 (Genetex, Irvine, CA, USA) overnight at 4 °C. After washing with TBST, the membranes were hybridized with anti-rabbit or anti-mouse IgG peroxidase-conjugated secondary antibody (Cell Signalling Technology) and developed by Western Chemiluminescent HRP Substrate (Millipore) using the ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA, USA). Blotted proteins were quantified using ImageLab 6.0.1 (BioRAd).
4
Intracellular ATP Quantification Assay
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The intracellular level of ATP was measured using ATP Colorimetric/Fluorometric Assay kit from BioVision (Mountain View, CA, USA) following the manufacturer’s instructions. Briefly, cells were treated with MS-5 (15, or 30 µM) for 6 h, 12 h and 24 h. Cells were lysed and centrifuged at 12,000 rpm for 5 min. The supernatant (100 µL) was transferred to a 24-well plate, and then mixed with ATP detection working solution (100 µL). Luminescence signals were measured by a microplate reader (BMG Labtech). The protein concentration of each group was also determined using a Coomassie protein assay reagent (Thermo scientific). The relative ATP level was expressed as ATP value/protein value.
5
Preparation of Crude Brain Membrane Fractions
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Crude human membranes from brain tissues were prepared as described previously [25 (link)]. Briefly, tissues were homogenized in 10 mM HEPES/KOH, pH 7.4; 0.32 M sucrose; 0.5 mM MgSO4; 0.1 mM phenylmethanesulfonyl fluoride (PMSF); 2 mM 2-mercaptoethanol; and protease inhibitor cocktail solution (Roche Diagnostics, Mannheim, Germany). The homogenates were first centrifuged at 1500 g for 10 min, and the supernatants were further centrifuged at 100,000 g for 45 min. The final pellets were resuspended in 10 mM HEPES/KOH, pH 7.4, 0.32 M sucrose. Protein content was determined using the Coomassie Protein Assay Reagent (Thermo Fisher Scientific).
6
HeLa and HEK293 Cell Lysis
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Human cervical carcinoma cells (HeLa) and HEK293 cells were obtained from BioWhittaker Europe and cultured as described previously [21 (link)]. HeLa cell was lysed in either buffer A or RIPA buffer (Thermo Scientific). Cell lysates were centrifuged at 14,000 g for 10 min at 4°C and the protein content of the supernatant determined using the Coomassie Protein Assay Reagent (Thermo Scientific) before normalisation and solubilisation in x2 SDS-PAGE sample buffer. Soluble and chromatin protein fractions were prepared as described previously [22 (link)].
7
Protein Extraction and Western Blotting
8
Pancreatic Hormone Content Quantification
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Whole pancreases obtained from the WT, A0B2, and A0B0 mice were weighed and chopped into small pieces in 6 ml ice-cold acid-ethanol (1.5% HCl in 75% ethanol). The tissues were then sonicated for 30 s (duty cycle, 20%; output control, 20) and stored overnight at 4°C, followed by a second round of sonication the next day. The supernatants were collected after centrifuging the samples at 2,400 rpm for 30 min at 4°C. The total protein concentration was measured with a Coomassie protein assay reagent (Thermo Scientific). Insulin and glucagon contents were measured by enzyme-linked immunosorbent assay (ELISA) (Morinaga mouse insulin ELISA kit [M1102]; Mercodia glucagon 10-μl ELISA kit [10-1281-OD]). Each pancreatic content was normalized by the total protein concentration per sample.
9
Isolation and Characterization of OMVs and Exosomes
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E. coli O6:K2:H1 (ATCC, Manassas, VA, USA) were cultured overnight in Luria-Bertani medium at 37 °C rotator with speed 180 rpm until the desired OD600 was reached. The cultured E. coli were then pelleted and filtered as previously described [22 (link)], followed by OMV purification using ExoBacteria OMV Isolation Kits (SBI, Palo Alto, CA, USA), based on the manufacturer’s protocol. The protein content in the isolated OMVs was measured using Bradford assays with Coomassie Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Exosomes were isolated from the bronchoalveolar lavage fluid (BALF) of WT mice. To obtain BALF, a total of 2 mL of PBS was used for the collection, as previously described [23 (link)]. BALF was centrifuged at 300× g for 10 min to remove floating cells, followed by 2000× g for 20 min to separate apoptotic bodies, and 16,000× g for 40 min to separate microvesicles [24 (link)]. The resulting supernatants were then ultracentrifuged at 100,000× g for 1 h to obtain exosomes [24 (link)]. TEM images of OMVs were generated at the Experimental Pathology Laboratory Core, Boston University School of Medicine.
10
Yeast Protein Expression and Detection
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Yeast strains GCY256 and GCY257 (Table 1 ) expressing HA-tagged PsSAS and PsCPR respectively, were grown overnight in SC medium with 0.2% glucose and 1.8% galactose as carbon sources. Ten ml of fresh medium containing 2% galactose as a carbon source was inoculated with 5% of the overnight cultures and incubated at 30°C and 200 rpm. Cells were harvested by centrifugation at OD600 of approximately 0.6 and lysed by bead beating in IP150 buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM MgCl2, 0.1% Nonidet P-40) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche Applied Science). The lysates were cleared by centrifugation and protein concentration was estimated using a Coomassie protein assay reagent (Thermo Scientific). Forty μg of total protein extract were resolved by SDS-PAGE and transferred to nitrocellulose for detection of the HA epitope using mouse anti-HA tag antibody HA-C5 (Abcam). As a loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected using rabbit anti-GAPDH (Rockland Immunochemicals). Imaging of the blot was performed using an Odyssey imager and IRDye secondary antibodies (LI-COR Biosciences).
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