Cells and clinical biopsies were lysed in a radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich; #11836170001). Protein concentrations in total cell extracts were quantified using the Pierce Coomassie Plus Assay Reagent (ThermoFisher Scientific; #23238). Equal amounts of total protein were loaded onto 12% SDS-PAGE gels. After transferring to PVDF (polyvinylidene fluoride) membranes (ThermoFisher Scientific; #PB9320) and blocking with 5% (w/v) nonfat milk, the proteins were detected by specific antibodies, including anti-CtIP (Santa Cruz Biotechnology; #sc-271339), anti-CtBP1 (Cell Signaling Technology, Shanghai, China; #8684), anti-CtBP2 (Cell Signaling Technology; #13256), anti-HDAC1 (Cell Signaling Technology; #34589), anti-JUN (Abcam; #ab40766), anti-FOS (Abcam; #ab134122), anti-Myc (Santa Cruz Biotechnology; #sc40), anti-Flag (Sigma-Aldrich; #F3165), anti-Actin (Abcam; #ab179467), anti-LSD1 (Abcam; #aab37165), and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Sigma-Aldrich; #G9545).
Pierce coomassie plus assay reagent
Manufactured by Thermo Fisher Scientific
Sourced in Lithuania
The Pierce Coomassie Plus Assay Reagent is a colorimetric protein quantitation solution used for the measurement of protein concentration in biological samples. It provides a simple, fast, and accurate method for determining total protein content.
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Lab products found in correlation
Accuskan, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Anti hdac1, by Cell Signaling Technology (1 mentions)
Anti actin, by Abcam (1 mentions)
Anti fos, by Abcam (1 mentions)
Anti lsd1, by Abcam (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
Anti ctbp2, by Cell Signaling Technology (1 mentions)
Anti ctip, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
E coli dh5α, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity pcr master mix, by Thermo Fisher Scientific (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Tskgel g4000swxl, by Tosoh (1 mentions)
M per buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Pronase, by Roche (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
5 protocols using pierce coomassie plus assay reagent
1
Western Blot Analysis of Cellular Proteins
2
Outer Membrane Vesicle Protein Quantification
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A Bradford assay was used to determine outer membrane vesicle protein concentrations, as previously described (20 (link)). To generate a standard curve, bovine serum albumin (BSA) was diluted at 0 to 20 mg/mL in Pierce Coomassie Plus assay reagent (Thermo Fisher) to a final volume of 1 mL. Outer membrane vesicles were diluted at 2, 5, 10, 15, and 20 μL in reagent to a final volume of 1 mL. A microplate spectrophotometer (Fisherbrand AccuSkan) was used to measure the absorbance (OD595) of the standard and samples in a 96-well plate (BrandTech). Protein concentrations were determined by comparing the optical densities of the samples to the standard curve plotted in Microsoft Excel, and final quantifications were graphed in GraphPad Prism 9. Experiments were reproduced three times from each outer membrane vesicle isolation, and one representative data set is reported.
3
Recombinant Protein Expression Workflow
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Restriction endonucleases (HindIII, XhoI, NcoI, SnaBI, NheI), T4 DNA ligase, Phusion High-Fidelity PCR Master Mix, PageRulerTM Prestained Protein Ladder, and Pierce™ Coomassie Plus Assay Reagent were purchased from Thermo Fisher Scientific, Vilnius, Lithuania. pET21d and pET28b vectors were purchased from Novagen, Madison, WI, USA. Nutrient medium was purchased from Roth, Karlsruhe, Germany. Sucrose and salts were purchased from Sigma-Aldrich, Buchs, Switzerland. TSKgel G4000SWXL was obtained from Tosoh Bioscience, Tokyo, Japan. E. coli DH5α (Thermo Fisher Scientific, Vilnius, Lithuania) and BL21 (DE3) (Novagene, Madison, WI, USA) were used for cloning and expression experiments.
4
Pronase-based Target Engagement Assay
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Target engagement was determined using DARTS assay as previously described16 (link),26 (link),40 . SW620 cells in logarithmic growth phase were collected by trypsinization and lysed 10 min on ice in M-PER buffer (Thermo Fisher Scientific) with protease inhibitors (Roche). Lysates were centrifuged at 16,000g, 4 °C for 10 min. Protein concentration was determined by Bradford method with Pierce Coomassie Plus Assay Reagent (Thermo Fisher Scientific). Samples were diluted in 1× TN buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl) and treated as indicated for 30 min at room temperature. Treated cell lysates were aliquoted into 20 µg protein aliquots and digested with pronase (Roche) at the indicated pronase to total protein ratios for 30 min at room temperature. Reaction was stopped by 4× Laemmli sample buffer (BioRad) with 100 mM dithiothreitol and boiling at 95 °C for 5 min. Protein stabilization was analysed by western blot.
For target engagement in intact cells, SW620 cells were treated in culture with indicated compounds for 3 h before collection, lysis and pronase treatment performed as described above.
For target engagement in intact cells, SW620 cells were treated in culture with indicated compounds for 3 h before collection, lysis and pronase treatment performed as described above.
5
Quantifying Outer Membrane Vesicle Proteins
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Bradford assay was used to determine outer membrane vesicle protein concentration, as previously described 44 (link) . To generate a standard curve, bovine serum albumin (BSA) was diluted 0 to 20 mg/mL in Pierce Coomassie Plus assay reagent (ThermoFisher) to a final volume of 1 mL. Outer membrane vesicles were diluted 2, 5, 10, 15, 20 L in reagent to a final volume of 1 mL. A microplate spectrophotometer (Fisherbrand AccuSkan) was used to measure the absorbance (OD595) of standard and samples in a 96-well plate (BrandTech). Protein concentrations were determined by comparing the optical densities of samples to the standard curve plotted in Microsoft Excel and final quantifications were graphed in GraphPad Prism 9. Experiments were reproduced three times from each outer membrane vesicle isolation, and one representative data set was reported.
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