Total carbohydrate levels of crude oyster mushroom extract were measured using the total Carbohydrate Quantification Assay Kit (Abcam, United Kingdom), according to the kit instructions. Total protein content of crude extract was also determined by Bradford assay [9 (link),10 ] using Bradford reagent (AppliChem, Darmstadt, Germany), according to manufacturer’s instructions with bovine serum albumin for a standard protein.
Bradford reagent
Manufactured by AppliChem
Sourced in Germany
The Bradford reagent is a colorimetric assay used for the quantitative determination of protein concentration. It is a dye-binding assay that utilizes the binding of Coomassie Brilliant Blue G-250 dye to proteins, resulting in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Total carbohydrate quantification assay kit, by Abcam (1 mentions)
Extraction buffer, by Thermo Fisher Scientific (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Rabbit anti cdc42, by Cell Signaling Technology (1 mentions)
Ecl chemiluminescence reagent, by GE Healthcare (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Supersignal west femto maximum sensitivity substrate ecl, by Thermo Fisher Scientific (1 mentions)
5 protocols using bradford reagent
1
Quantifying Carbohydrates and Proteins in Oyster Mushroom Extract
2
Protein Quantification in Liver and Kidney
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The total protein content of liver and kidney homogenates was evaluated spectrophotometrically at 594 nm using Bradford Reagent (Applichem®, Darmstadt, Germany) and bovine serum albumin (BSA) as standard. The protein content was quantified to normalize the results obtained for the enzymatic activities.
3
Quantifying Microalgal Protein Content
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Total protein content was estimated on aliquots of 30 mg of dried microalgal powder re-suspended in 500 μL of RIPA (Radio-Immunoprecipitation Assay) and Extraction Buffer (Thermo Fisher Scientific, Waltham, MA, USA) and sonicated for 90 s (30 s on, 30 s off, 30 s on) using a micro tip at 20% output on ice (S-250A Branson Ultrasonic). Samples were then centrifuged at 13,000×g for 4 min, and supernatants were collected for protein content estimation. For this, 20 μL of sample were added into a 96 well plate (transparent flat bottom, TPP Techno Plastic Products AG, Trasadingen, Switzerland), with then the addition of 200 μL of Bradford reagent (A6932, AppliChem GmbH, Darmstadt, Germany). The absorbance was read at 595 nm, using a Microplate Reader: Infinite® M1000 PRO (TECAN, Männedorf, Switzerland). The total protein concentration was quantified referring to a calibration curve using bovine serum albumin (BSA) as standard.
4
Western Blot Analysis of Signaling Proteins
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Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCL [pH 7.6], 150 mM NaCl, 1% NP40, 1% sodium deoxycholate [SDS], plus protease inhibitor cocktail set III [Calbiochem, Darmstadt, Germany]). Protein concentration of the lysates was quantified by using Bradford reagent (Applichem). Samples were separated by SDS-Page and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were probed with rabbit anti-ITK (ab32113, Abcam, Cambridge, United Kingdom, 1:2000), goat anti-GAPDH (EB06377, Everest Biotech, 1:20,000), rabbit anti-CDC42 (#24645, Cell Signaling, Cambridge, United Kingdom, 1:500) or mouse anti-RAC1 (88751, BD Biosciences, 1:500) followed by horseradish peroxidase-conjugated rabbit anti-mouse antibody or goat anti-rabbit antibody (GE Healthcare) or rabbit anti-goat antibody (R131HRP, Acris), and developed with ECL chemiluminescence reagents (GE Healthcare).
5
Western Blot Analysis Protocol
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Cells were washed twice in PBS, lysed in modified RIPA buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.1% Na-deoxycholate, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail, 5 mM β-glycerophosphate, 5 mM NaF, 1 mM Na-orthovanadate, 10 mM N-ethylmaleimide), and centrifuged at 15,000×g for 15 min. at 4°C. Protein concentrations were estimated using Bradford Reagent (AppliChem).
Protein extracts were combined with 4× Laemmli Sample Buffer (Bio-Rad), boiled for 10 min at 70°C, resolved on MiniPROTEAN TGX pre-cast 4–15% SDS-polyacrylamide gels (10- or 15-well), and transferred onto Hyperfilm enhanced chemiluminescence (ECL) nitrocellulose membranes (GE Healthcare). Following the transfer, membranes were blocked with 5% milk in TBST, and probed with primary and secondary antibodies. For visualization SuperSignal West Femto Maximum Sensitivity ECL Substrate (Thermo Fisher Scientific) and an Odyssey imaging system (LI-COR Biosciences) were used.
Protein extracts were combined with 4× Laemmli Sample Buffer (Bio-Rad), boiled for 10 min at 70°C, resolved on MiniPROTEAN TGX pre-cast 4–15% SDS-polyacrylamide gels (10- or 15-well), and transferred onto Hyperfilm enhanced chemiluminescence (ECL) nitrocellulose membranes (GE Healthcare). Following the transfer, membranes were blocked with 5% milk in TBST, and probed with primary and secondary antibodies. For visualization SuperSignal West Femto Maximum Sensitivity ECL Substrate (Thermo Fisher Scientific) and an Odyssey imaging system (LI-COR Biosciences) were used.
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