The biomass of T. reesei grown in TMM + 2% cellulose with and without glutamine was indirectly determined by the amount of intracellular protein [63 (link), 64 (link)]. In brief, harvested mycelia were suspended in 1 M NaOH and incubated for 2 h with frequent vortex. Then the protein concentration of the supernatant of the suspension was determined by the Modified BCA Protein Assay Kit (Sangon Biotech, Shanghai, China). The biomass dry weight was calculated assuming an average content of 0.32 g intracellular protein per g of dry cell mass.
Modified bca protein assay kit
Manufactured by Sangon
Sourced in China
The Modified BCA Protein Assay Kit is a colorimetric assay used to quantify the total protein concentration in a sample. It is based on the bicinchoninic acid (BCA) method, which uses a copper-based reagent to produce a purple-colored complex with proteins. The kit includes the necessary reagents and solutions to perform the assay, and provides a simple and reliable way to measure protein levels in a variety of biological samples.
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Lab products found in correlation
Pd 10 desalting column, by GE Healthcare (2 mentions)
Bl21 de3 plyss, by Promega (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Rabbit anti flag, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti cldnd1, by Abcam (1 mentions)
Ds 11, by DeNovix (1 mentions)
Hipure bacterial rna kit, by Magen Biotechnology Co (1 mentions)
Bacterial protein extraction kit, by Sangon (1 mentions)
Uv 6000 spectrophotometer, by Metash (1 mentions)
Chemiluminescent emsa kit, by Beyotime (1 mentions)
Nuclear extract kit, by Beyotime (1 mentions)
X omat bt x ray film, by Kodak (1 mentions)
Glutamic acid measurement kit, by Nanjing Jiancheng (1 mentions)
Glutamine measurement kit, by Nanjing Jiancheng (1 mentions)
Synergy h4, by Agilent Technologies (1 mentions)
Vibra cell, by Sonics (1 mentions)
Cytoplasmic protein extraction kit, by Beyotime (1 mentions)
10 protocols using modified bca protein assay kit
1
Measuring Trichoderma reesei Biomass
2
Western Blot Analysis of CLDND1 Expression
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Cells in 6-well cell culture plate were lysed using 150 μL RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors. Then cell lysates were collected and centrifuged at 10,000 rcf and 4℃ for 15 minutes. The protein concentrations were measured using the Modified BCA Protein Assay Kit (Sangon Biotech). After mixing and boiling with the SDS-PAGE loading buffer, 40 μg of total protein was electrophoresed in 10% SDS-PAGE gels and transferred on to a PVDF membrane (Merck Millipore). Then the PVDF membrane was blocked in PBST buffer supplemented with 5% skim milk and 0.1% Tween 20 for 1 hour at room temperature and incubated with the primary antibodies overnight at 4℃. After incubation with the consistent HRP-conjugated secondary antibodies, the protein bands were detected using SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The following antibodies were used according to the manufacturers' instructions: rabbit anti-CLDND1 (Abcam, MA, USA), rabbit anti-Flag (Sigma-Aldrich), mouse anti-β-actin (CMCTAG, WI, USA), HRP-labeled anti-rabbit IgG (KPL, MD, USA), and HRP-labeled anti-mouse IgG (KPL).
3
Cloning and Purification of CrOMT1 Protein
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The CrOMT1 gene was isolated based on the C. clementina reference genome. The full coding sequence (CDS) of CrOMT1 was amplified using the primers described in Supplementary Table S1 and then subcloned into the pET32a vector (without a stop codon) followed by transfer to E. coli strain BL21(DE3)pLysS (Promega, Madison, WI, USA) for expression. Transformants carrying the expression plasmid CrOMT1-PET were incubated to OD600=0.6 in Luria–Bertani medium containing 0.1 g l–1 ampicillin at 37 °C, followed by the addition of isopropyl-β-d -thiogalactopyranoside (IPTG) to a final concentration of 1 mM and then the incubation was carried out for 24 h at 18 °C. After induction, the bacterial cells were collected (4000 rpm, 4 °C, 10 min) and resuspended in 1× PBS buffer. The clarified supernatant of cells disrupted by sonication was obtained by centrifugation at 4 °C and the corresponding recombinant proteins were purified using a HisTALON™ Gravity Columns Purification Kit (Takara, Dalian, China) according to the user manual. Collected fractions were transferred into storage buffer (50 mM Tris–HCl, pH 8.0, 10% glycerol, and 2 mM DTT) through a PD-10 desalting column (GE Healthcare, Uppsala, Sweden), and stored at –80 °C for further analysis. Protein concentrations were determined via the Modified BCA Protein Assay Kit (Sangon Biotech, Shanghai, China).
4
Bacterial Protein and RNA Extraction
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The culture was sampled every 6 h, and then concentrated or diluted to 1 (OD600). Protein was extracted using Bacterial Protein Extraction Kit (BS596, Sangon Biotech Co, Ltd, Shanghai, China) and quantified by a Modified BCA Protein Assay Kit (SK3051; Sangon Biotech Co, Ltd, Shanghai, China). Total RNA was extracted by HiPure Bacterial RNA Kit (R4181-01; Magen, China) and quantified by a spectrophotometer (DS-11; DeNovix, Wilmington, DE, USA).
5
Characterization of MEC Solutions
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Chemical oxygen demand (COD) of solution in MECs after a whole cycle was measured after three steady fed-batch cycles according to the standard methods of American Public Health Association [46 ]. VFAs were analyzed by gas chromatograph (GC4890, Agilent, America). Protein concentration was gauged by UV-6000 spectrophotometer (METASH, China) with Modified BCA Protein Assay kit (Sangon Biotech, China). The content of polysaccharides was detected by phenol-vitriol colorimetry method [47 ]. Samples for VFAs, soluble COD, soluble protein and soluble polysaccharides characterization were obtained by filtering with 0.45 μm filter membrane.
6
Hnf4α Binding to elovl5 Promoter
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To confirm the binding of Hnf4α to the promoter of rabbitfish elovl5, nuclear and cytoplasmic proteins were extracted from rabbitfish hepatocytes with the Beyotime Nuclear Extract Kit (Beyotime Institute of Biotechnology, Haimen, China) and quantified by Modified BCA Protein Assay Kit (Sangon, Shanghai, China). The 29 bp 5′ end biotin-labeled probe covering the predicted Hnf4α elements was designed and incubated with the proteins to determine whether Hnf4α interacted with the promoter of elovl5. Both the labeled and unlabeled probes in the experiment were obtained from Shanghai Sangon Biotech Co., Ltd., while the EMSA reaction system was performed with the Beyotime Chemiluminescent EMSA Kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions. For the super shift assay, 1 μL antibody (Abcam, Cambridge, MA, USA) of Hnf4α was pre-incubated with nuclear or cytoplasmic proteins for 30 min at 0–4 °C. Samples obtained after the binding reaction were subjected to a 4% non-denaturing polyacrylamide gel electrophoresis and transferred onto a nylon membrane. The 5′ end biotin-labeled probe was detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The signal was then detected by autoradiography with X-OMAT BT X-ray film (Kodak, Rochester, MN, USA).
7
Nitrogen Metabolism Enzyme Assay
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N metabolism in plants is closely associated with the activities of several key enzymes, such as NR and GS [62 (link)]. For NR activity determination, the roots and leaves harvested were frozen in liquid N immediately, and then stored at − 80 °C until further analysis. Samples were ground to a fine powder (~ 100 mg), extracted, and analyzed spectrophotometrically [57 (link), 63 , 64 (link)]. The activity of GS was assayed as reported by Wang et al. [65 (link)]. The activity of NADH-GOGAT was quantified using a NADH-GOGAT measurement kit (Solarbio Bioengineering Institute). Glutamate and glutamine were quantified using a glutamic acid measurement kit and a glutamine measurement kit, respectively (both from Nanjing Jiancheng Bioengineering Institute). Enzyme activities were expressed as moles of metabolite generated/consumed per milligram of fresh weight or protein per unit of time. The protein concentration was determined by the Coomassie brilliant blue method with Modified BCA Protein Assay Kit, C503051, Sangon Biotech.
8
Heterologous Expression and Purification of CsCCoAOMT1
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The CsCCoAOMT1 gene was isolated on the basis of C. sinensis reference genome. The full coding sequence of CsCCoAOMT1 was inserted into the pET32a vector using the primers in Supplemental Table S3. After sequence validation, CsCCoAOMT1-pET32a construct was transferred to E. coli strain BL21 (DE3) pLysS (Promega, Madison, WI, USA) for expression. The recombinant protein was expressed and purified as described by Liu et al. (2020b) : purified by the HisTALON Gravity column (Takara) and replaced with Tris-HCl storage buffer (50 mM, pH 8.0) by the PD-10 Desalting Column (GE Healthcare, Uppsala, Sweden). The purified protein was stored at -80 °C and the concentration was measured using a Modified BCA Protein Assay Kit (Sangon Biotech, Shanghai, China).
9
Quantifying Trichoderma reesei Biomass
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The biomass of T. reesei grown in TMM + 2% cellulose was indirectly determined by the amount of intracellular protein [46 (link), 47 (link)]. In brief, the mycelia of T. reesei were suspended in 1 M NaOH and incubated for 2 h with frequent vortex. Then the protein concentration of the supernatant of the suspension was determined by the Modified BCA Protein Assay Kit (Sangon Biotech, Shanghai, China). The biomass dry weight was calculated assuming an average content of 0.32 g intracellular protein per g of dry cell mass.
10
Membrane and Cytoplasmic Protein Extraction
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The strains SSAAI-10, SSAAI-egfp, and SSAAI-serE-egfp were used for extracting membrane and cytoplasmic proteins to determine SerE localization. The extraction was performed using Membrane and a Cytoplasmic Protein Extraction Kit according to the manufacturer’s protocol (Beyotime, Nanjing, China). The cells were washed twice with PBS (pH 7.4) and disrupted by ultrasonication on ice (pulse, 4 s; interval, 6 s; total duration, 30 min) (Sonics Vibra-Cell™, Sonics, Newtown, CT, USA). The supernatant containing cytoplasmic proteins was collected by centrifugation (700×g, 4 °C for 10 min), and the precipitate was used for extracting membrane proteins. The protein concentration was determined with a Modified BCA Protein Assay Kit (Sangon, China). After extraction, the fluorescence intensity (excitation at 488 nm, emission at 517 nm) of the membrane and cytoplasmic proteins was determined using fluorescence spectrophotometer (Synergy H4; BioTek, Winooski, VT, USA).
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