HeLa cells were seeded into 12-well plates (150,000 cells/well) and transfected with the indicated plasmids using polyethylenimine (PEI, Polysciences). Twenty-four hours after transfection cells were washed in 1 ml 1X PBS (Sigma) and lysed in 150 μl 1X Passive Lysis Buffer (Promega) for 10 min with rocking at room temperature. After lysis supernatants were cleared of debris by centrifugation at 10,000 x g for 1 min. For firefly luciferase, 40 μl of luciferase reagent (Promega) was added to 8 μl of sample, and luciferase activity was measured using a luminometer (Molecular Devices). For experiments using bicistronic reporters, 40 μl of Stop and Glo reagent was added to each sample after measuring firefly luciferase levels. 40 μl of renilla luciferase reagent (Promega) was then added, and luciferase activity was again measured. The amount of luciferase activity was normalized to the amount of protein present in each sample as determined by Bradford assay (Amresco).
Bradford assay
Manufactured by Avantor
Sourced in United States
The Bradford assay is a colorimetric protein quantification method. It is used to determine the concentration of protein in a solution.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitors cocktail, by Thermo Fisher Scientific (2 mentions)
M per lysis buffer, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Renilla luciferase reagent, by Promega (1 mentions)
Luciferase reagent, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Ab3786, by Merck Group (1 mentions)
Lc3b rabbit polyclonal antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Microchemi 4.2 system, by DNR Bio-Imaging Systems (1 mentions)
Chemidoc mp imaging station, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Rabbit anti human gapdh antibody, by Abcam (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Microfuge, by Beckman Coulter (1 mentions)
Ab183628, by Abcam (1 mentions)
Supersignal west femto maximum sensitivity, by Thermo Fisher Scientific (1 mentions)
11 protocols using bradford assay
1
Measuring Luciferase Activity in Transfected Cells
2
Western Blot Analysis of PCNA
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Frozen tumor samples were minced and homogenized (Pro scientific, Oxford, CT, USA) in lysis buffer [120 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% NP-40 and complete protease inhibitor cocktail tablets (1 tablet/50 mL, cat. 11697498001; Roche Diagnostics, Indianapolis, IN, USA)]. After incubation on ice for 30 min, the tissue homogenates were sonicated for 8–10 pulses at 80% amplitude. Lysates were then centrifuged for 15 min at 12,000× g and protein concentrations of the supernatants were determined by using the Bradford Assay (Amresco, cat. E530). Tumor tissue lysate (40 μg) was electrophoresed on a 4–12% gel, transferred to a nitrocellulose membrane, and blocked in 5% milk for two hours. To examine PCNA expression, membranes were incubated overnight at 4 °C with a PCNA antibody (BD Transduction laboratories, cat. 610665, 1:1000 dilution). Mouse secondary antibodies (1:10,000 dilution, cat. A24524; Invitrogen, Carlsbad, CA, USA) were incubated for one hour and the blots were developed using ECL (cat. 80196, Pierce ECL2 western blotting substrate) and exposed to film. Densitometry was performed on the blots using ImageJ analysis software 1.50i (National Institutes of Health, Bethesda, MD, USA).
3
Lung Tissue Protein Extraction and Western Blot
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Frozen lung tissues were sonicated in RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 1 mM EDTA) containing protease inhibitor cocktail (Roche Applied Bioscience). Protein content was determined by Bradford assay (Amresco) staining using bovine serum albumin as a standard. Equal amounts of protein were loaded on 10%, 12%, or 15% Tris-glycine gels for electrophoresis. Proteins were wet-transferred to PVDF membranes (Millipore) and then probed with the indicated antibodies. The primary antibodies and dilutions used were as follows: anti-surfactant protein B rabbit polyclonal antibody (1:3,000, #07-614, Millipore), anti-surfactant protein C rabbit polyclonal antibody (1:1000, #AB3786, Millipore), anti-surfactant protein A rabbit polyclonal antibody (1:1000, #AB3420, Millipore), anti-p62 rabbit polyclonal antibody (1:1000, #P0067, Sigma), LC3B rabbit polyclonal antibody (1:1000, #12741, Cell Signaling Technology), anti-ATG7 rabbit polyclonal antibody (1:1000, #A2856, Sigma) and anti-actin mouse polyclonal antibody (1:1000, #TA-09, Zsgb-bio, China). Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies. Chemiluminescence substrates were used (Tiangen, Beijing, China). The images were captured using a MicroChemi 4.2 system (DNR Bio Imaging Systems, Jerusalem, Israel).
4
Protein Extraction and Characterization
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Proteins were extracted using the Protominer kit (BioRad, Hercules, CA, USA), and protein concentration and sample integrity were assessed by the Bradford assay (Amresco, Burlington, NC,
USA), and polyacrylamide gel electrophoresis (BBI, Shanghai, China).
USA), and polyacrylamide gel electrophoresis (BBI, Shanghai, China).
5
Purification and Characterization of CbpA Variants
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CbpAs used in this study were CbpA2, CbpA3 and variants thereof, CbpA5, and CbpA6. CbpAs2,3,5,6 were expressed and purified as previously published (16 ) (see Supplemental Text for details). Protein purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) analysis (Fig. S10A–E ), visualized with Coomassie blue staining using a ChemiDoc MP Imaging Station (Bio-Rad). Concentrations of CbpAs and variants thereof were determined using absorbance readings at 280 nm, employing extinction coefficients and molecular weights calculated with Expasy (Table S4 ). The concentration of CbpA396–283 was estimated with a Bradford assay (Amresco), using bovine serum albumin as a calibration standard.
6
Quantification of Nascent Protein Synthesis
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Nascent proteins
were metabolically labeled and quantified as described previously.25 (link),26 (link) Briefly, cells were incubated in methionine- and cysteine-free medium
(Sigma) containing relevant concentrations of compounds tested for
15 min. 35S-labeled methionine and cysteine (125 μCi;
PerkinElmer EasyTag Express Labeling Mix) were added and allowed to
incorporate for 30 min. Cells were then washed twice in ice-cold PBS,
scraped, and collected by centrifugation. Cell pellets were lysed
in RIPA medium containing protease inhibitors (Roche), and protein
concentrations were determined by the Bradford assay (Amresco). Trichloroacetic
acid (TCA) was added to a final concentration of 20%, and precipitated
proteins were captured on glass microfiber filters by filtration under
vacuum. The filters were washed twice with 20% TCA and once with 100%
ethanol and allowed to air-dry. The filters were then transferred
to vials containing scintillation fluid (EcoScint), and radioactivity
was quantified using a scintillation counter. The amount of radioactivity
was normalized to the protein concentration for each sample.
were metabolically labeled and quantified as described previously.25 (link),26 (link) Briefly, cells were incubated in methionine- and cysteine-free medium
(Sigma) containing relevant concentrations of compounds tested for
15 min. 35S-labeled methionine and cysteine (125 μCi;
PerkinElmer EasyTag Express Labeling Mix) were added and allowed to
incorporate for 30 min. Cells were then washed twice in ice-cold PBS,
scraped, and collected by centrifugation. Cell pellets were lysed
in RIPA medium containing protease inhibitors (Roche), and protein
concentrations were determined by the Bradford assay (Amresco). Trichloroacetic
acid (TCA) was added to a final concentration of 20%, and precipitated
proteins were captured on glass microfiber filters by filtration under
vacuum. The filters were washed twice with 20% TCA and once with 100%
ethanol and allowed to air-dry. The filters were then transferred
to vials containing scintillation fluid (EcoScint), and radioactivity
was quantified using a scintillation counter. The amount of radioactivity
was normalized to the protein concentration for each sample.
7
Immunoblotting for PDCD4 Protein
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Whole-cell extracts were prepared using M-PER lysis buffer (Pierce). The concentration of proteins was measured by Bradford assay (Amresco), and equal protein amounts were used for SDS-PAGE. Briefly, proteins from whole-cell extracts were separated and transferred onto nitrocellulose membranes. The membranes were blocked with 1× TBS-T with 5% nonfat dry milk (Bio-Rad), followed by incubation overnight at 4°C with 1:1000-diluted primary anti-rabbit PDCD4 antibody (Cell Signaling). For protein loading controls, rabbit anti-human GAPDH antibody (Abcam) was used at 1:2500 dilution in Superblock T20 buffer (Pierce). The blot was then probed with 1:10,000-diluted goat anti-rabbit IgG secondary-HRP antibody (Pierce). The signal was detected by Super Signal West Femto ECL (Pierce).
8
Osteoblast Protein Extraction Protocol
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Osteoblasts cells were incubated in 50 mL culture flasks and grown to subconfluence (approximately 60%–70%) and then treated with test (HQSXD-S) and control (JKSQW-control-S, BLANK-control-S) at a concentration of 10% (v/v) for 72 h, respectively. At the end of the incubation period, osteoblasts were collected by centrifugation at 1,000 rpm (4°C). The osteoblasts were washed twice with cold PBS, after discarding the medium. Total proteins were extracted in a chilled lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM dithiothreitol (DTT), 2% (v/v) IPG buffer, pH 3–10, 4 μg/mL protease inhibitor mixture, and 4 μg/mL phosphatase inhibitors. After addition of the chilled lysis buffer, the cell solution was kept oscillating for 1 h at 4°C to solubilize the proteins. The homogenate was subsequently centrifuged for 15 min at 14,000 rpm at 4°C. Total protein concentration was quantified using the Bradford assay (Amresco).
9
In Vitro TT-Loaded PCL Nanoparticle Stability
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Ten milligrams of TT-loaded PCL NPs (60 nm or 450 nm) was dissolved in 1 ml of either PBS (pH 7.2), PBS (pH 4.0), or double-distilled water (pH 6.0) and kept at 37°C for 7, 14, 21, 28, and 30 days. After the indicated time points, the Microfuge® (Beckman Coulter, Pasadena, CA, USA) tubes were centrifuged at 15,000 rpm, and the supernatants were assayed for protein content using a Bradford assay (Amresco, Solon, OH, USA). Values were obtained from the standard curve obtained with bovine serum albumin (BSA) as reference protein. Void PCL NPs were used as negative controls.
10
WES Protein Analysis of Cell Lysates
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The protein levels in cells were determined by WES protein analysis (ProteinSimple, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, cells were washed with ice-cold PBS and lysed in MPER lysis buffer (Thermo Fischer Scientific) containing protease inhibitors cocktail (Thermo Fischer Scientific) by incubating for 15 min on ice. The cell debris was removed by centrifugation at 10,000× g for 15 min at 4 °C. The protein levels in clear cell lysate were measured by Bradford assay (VWR Life Science). Total protein (250 or 500 ng) was separated and immunoprobed in a capillary system using the WES ProteinSimple system: rabbit polyclonal antibody for mCherry (Abcam ab183628) and rabbit monoclonal antibody for GAPDH (Cell Signaling #5174) were used at 1:50 or 1:100 dilutions. Quantitative results such as molecular weight and signal intensity (area) were obtained using the WES ProteinSimple system software according to the manufacturer’s instructions.
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