Aluminum chloride (AlCl3), Folin-Ciocâlteu reagent, indomethacin, carboxymethylcellulose, o-phthalaldehyde, Lambda carrageenan type IV, sodium carbonate (Na2CO3), ABTS (diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate), DPPH (2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl), and TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), were purchased from Sigma–Aldrich (Taufkirchen, Germany). 2-thiobarbituric acid and Bradford reagent were obtained from Merck KGaA (Darmstadt, Germany) and ELISA tests for cytokines (TNF-α, IL-6) were purchased from Elabscience (Houston, TX, USA). The Bradford total protein assay was obtained from Biorad (Hercules, CA, USA). All HPLC reagents and standards were of analytical grade and were acquired from Sigma–Aldrich (Germany) and Decorias (Rediu, Romania), respectively.
Bradford total protein assay
Manufactured by Bio-Rad
Sourced in United States
The Bradford total protein assay is a colorimetric assay used to measure the total protein concentration in a sample. The assay is based on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, which results in a color change that can be detected and quantified spectrophotometrically. The Bradford assay provides a quick and simple method for determining the total protein content in a variety of sample types, including cell lysates, purified protein solutions, and biological fluids.
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Lab products found in correlation
Carboxymethylcellulose, by Merck Group (1 mentions)
Folin cioc lteu reagent, by Merck Group (1 mentions)
2 thiobarbituric acid, by Merck Group (1 mentions)
Lambda carrageenan type 4, by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
O phthalaldehyde, by Merck Group (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Irdye 680 goat anti mouse igg, by LI COR (1 mentions)
Odyssey ir scanner, by LI COR (1 mentions)
4 protocols using bradford total protein assay
1
Measurement of Antioxidant and Anti-Inflammatory Activities
2
Quantitative analysis of bacterial protein expression
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Bacteria were harvested from GC agar plates in PBS to achieve an OD600 = 1. The bacterial suspensions were normalized according to the total protein content, as determined using a Bradford total protein assay (Bio-Rad, Hercules, CI, USA), and equal amounts of total protein were separated using 12% SDS-PAGE. All samples were boiled in reducing sample buffer at 95°C for 5 min prior to electrophoresis. The proteins were transferred from the gel onto PVDF sheets and overlaid with primary antibodies: rabbit anti-PilT (1:10,000) and mouse anti-EF-Tu (1:2,000). Incubation with the primary antibodies was followed by two different fluorescent dye-conjugated secondary antibodies for the detection of PilT (goat anti rabbit IgG IRdye800CW (Li-COR)) (1:10,000) and for the detection of EF-Tu (goat anti-mouse IgG IRdye680 (Li-COR)) (1:20,000). The membrane was visualized and analyzed using an Odyssey IR scanner (Li-COR, Lincoln, NE, USA) at 700 and 800 nm. See the Figure legends for information concerning the number of repeats.
3
Exosome Isolation Protocol
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Exosomes were isolated as previously described by Thery et al.28 Briefly, cultures were centrifuged 10 min at 850 g. Supernatants were collected and centrifuged for 15 min at 3000 g to remove cell debris. Further, the supernatants were spun for 30 min at 10 000 g followed by filtration through 0.2-μm filter. Finally, the supernatants were ultracentrifuged for 70 min at 100 000 g to pellet the exosomes. The protein content of the exosome fraction was measured using Bradford total protein assay (Bio-Rad, Hercules, CA, USA).
4
ELISA for Bacterial Pilus Antibody Detection
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The ELISA was performed as described previously [42 (link)]. Briefly, the wells of a 96-well microtiter plate were coated with 50 μl of either a bacterial suspension in PBS (OD600 = 0.005) or diluted whole bacterial protein extracts. To prepare whole bacterial protein extracts, one ml of a bacterial suspension at OD600 = 0.32 in PBS was mixed with 100 μl of trichloroacetic acid, followed by a 15 min incubation on ice. After a 5 min centrifugation at 20,000× g, the pellet was dissolved in PBS and diluted before being loaded into a 96-well plate. Equal loading was determined using a Bradford total protein assay (Bio-Rad). The samples were allowed to adhere for 2 h and blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature. The samples were incubated for 1 h at 37°C and 5% CO2 with an anti-FAM20 pilus antibody (1:5,000) [38 (link)] and subsequently incubated with an HRP-conjugated anti-rabbit antibody (1:5,000) for another hour at 37°C. HRP was detected using 3, 3’, 5, 5’-tetramethylbenzidine (TMB), and the reaction was stopped using 1 M HCl. The absorbance at OD450 was read using a microplate reader. The experiment was performed twice in triplicate.
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