Biotin-conjugated rat anti-mouse IgE-paired antibodies were obtained from BD BioSciences (San Jose, CA, USA). Streptavidin alkaline phosphatase was obtained from Jackson ImmunoResearch (West Grove, PA, USA). BSA standard (at 2 mg/mL) was purchased from Sigma (St. Louis, MO, USA). p-nitro-phenyl phosphate was obtained from Sigma (St. Louis, MO, USA). Alkaline copper tartrate was purchased from BioRad (Hercules, CA, USA). Folin reagent was purchased from BioRad (Hercules, CA, USA). The following reagents were obtained from Invitrogen (Waltham, MA, USA): IgE Mouse Uncoated ELISA Kit with Plates; StreptAvidin-HRP, TMB substrate; MCPT-1 (mMCP-1) Mouse Uncoated ELISA Kit with Plates; Avidin-HRP, TMB substrate. Tissue Protein Extraction Reagent [T-PERTM, a proprietary detergent in 25 mM bicine and 150 mM sodium chloride (pH 7.6)] was from ThermoFisher Scientific (Waltham, MA, USA). A protease (serine, cysteine, acid proteases and aminopeptidases) inhibitor cocktail was obtained from Sigma-Aldrich (St. Louis, MO, USA).
Bsa standard
Manufactured by Merck Group
Sourced in United States
The BSA (Bovine Serum Albumin) Standard is a laboratory reagent commonly used as a protein standard in various analytical techniques. It serves as a reference to quantify the concentration of proteins in samples. The BSA Standard is made from a purified form of the bovine serum albumin protein and is available in different concentrations to meet the needs of different experimental protocols.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin hrp, by Thermo Fisher Scientific (2 mentions)
Streptavidin alkaline phosphatase, by Jackson ImmunoResearch (2 mentions)
Biotin conjugated rat anti mouse ige paired antibodies, by BD (2 mentions)
Alkaline copper tartrate, by Bio-Rad (2 mentions)
Folin reagent, by Bio-Rad (2 mentions)
Ige mouse uncoated elisa kit with plates, by Thermo Fisher Scientific (2 mentions)
Mcpt 1 mmcp 1 mouse uncoated elisa kit with plates, by Thermo Fisher Scientific (2 mentions)
Avidin hrp, by Thermo Fisher Scientific (2 mentions)
P nitrophenyl phosphate, by Merck Group (2 mentions)
Agilent 1100 series uv detector, by Agilent Technologies (2 mentions)
Superose6 10 300 gl, by Merck Group (2 mentions)
Dawn heleos 8 light scattering detector, by Wyatt Technology (2 mentions)
Agilent 1200 series refractive index detector, by Agilent Technologies (2 mentions)
Protease inhibitor cocktail tablets, by Abcam (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Direct detect system, by Merck Group (1 mentions)
N acetyl d galactosamine, by Merck Group (1 mentions)
L fucose, by Merck Group (1 mentions)
Biophen fviii c kit, by Hyphen Biomed (1 mentions)
Millipore milli q plus system, by Merck Group (1 mentions)
13 protocols using bsa standard
1
ELISA Protocol for IgE and MCPT-1
2
Quantitative Infrared Analysis of LiPs
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Infrared spectroscopy using the Direct Detect® system (EMD Millipore) was used to quantitatively determine the concentration of protein and lipids in the LiPs. The instrument uses a calibration via a BSA standard (Sigma) to quantify the protein abundance at multiple wavenumbers, including 1650 cm−1. POPG lipid signal was calibrated manually using several dilutions of POPG in Na-cholate buffer. Signal from the C-H symmetric stretching vibrational populations between 2870 and 2840 cm−1 was used to quantify lipid signals (see supplementary Fig. S1 for data and more information).
3
Characterization of Sea Cucumber Oligosaccharides
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Dried sea cucumbers H. fuscopunctata Jaeger were purchased from local markets in Guangdong Province and Hainan Province, China. Five oligosaccharide standards from LFG-Na (HS5, pentasaccharide, = 1506 Da; HS8, octasaccharide, = 2462 Da; HS11, hendecasaccharide, = 3417 Da; HS14, tetradecasaccharide, = 4373 Da; HS17, heptadecasaccharide, = 5328 Da) were obtained from Kunming Institute of Botany, Chinese Academy of Sciences. BSA standard was purchased from Sigma (St. Louis, MO, USA). The monosaccharides including L-fucose and N-acetyl-D-galactosamine were purchased from Sigma-Aldrich, and D-glucuronic acid was from J&K Scientific Ltd. (Beijing, China). 1-Phenyl-3-methyl-5-pyrazolone (PMP) was purchased from Xiya Reagnent Co. (Linyi, China). Amberlite FPA98Cl ion-exchange resin was purchased from Rohm and Haas Company (Philadelphia, PA, USA). Deuterium oxide (D2O, 99.9% Atom D) was obtained from Sigma-Aldrich. The activated partial thromboplastin time (APTT) kits, CaCl2 and standard human plasma were purchased from MDC Hemostasis (Neufahrn, Germany), BIOPHEN FVIII: C kit was from Hyphen Biomed (Neuville sur Oise, France). Human factor VIII was from Bayer HealthCare LLC. Deionized water was prepared by the Millipore Milli Q-Plus system (Millipore, Billerica, MA, USA). All other reagents were of analytical grade and obtained commercially.
4
Rat anti-mouse IgE ELISA protocol
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Biotin-conjugated rat anti-mouse IgE-paired antibodies were purchased from BD BioSciences (San Jose, CA, United States). p-Nitro-phenyl phosphate was obtained from Sigma (St Louis, MO, United States). Streptavidin alkaline phosphatase was obtained from Jackson ImmunoResearch (West Grove, PA, United States). BSA standard (at 2 mg/ml) was purchased from Sigma (St. Louis, MO, United States). Alkaline copper tartrate was purchased from BioRad (Hercules, CA, United States). Folin reagent was purchased from BioRad (Hercules, CA). The following reagents were obtained as listed: IgE Mouse Uncoated ELISA Kit with Plates; StreptAvidin-HRP, TMB substrate; MCPT-1 (mMCP-1) Mouse Uncoated ELISA Kit with Plates; Avidin-HRP, TMB substrate (all from Invitrogen, MA, United States); Tissue Protein Extraction Reagent (T-PER™, a proprietary detergent in 25 mM bicine, 150 mM sodium chloride, pH 7.6; from ThermoFisher Scientific, MA, United States); protease (serine, cysteine, and acid proteases, and aminopeptidases) inhibitor cocktail (Sigma-Aldrich, MO, United States).
5
SEC-MALS Analysis of Protein Samples
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Samples analyzed by SEC-MALS were buffer-exchanged by dialysis into SEC-MALS running buffer containing 20 mm HEPES (pH 7.6), 200 mm NaCl, 5% glycerol, 0.5 mm TCEP, and 0.05% azide. The samples were applied to a Superose6 10/300 GL (Sigma–Aldrich) column, and in-line measurements were recorded for UV absorbance at 280 nm (using Agilent 1100 series UV detector, Agilent Technologies), static light scattering (DAWN HELEOS 8+light scattering detector, Wyatt Technology), and differential refractive index (Agilent 1200 series refractive index detector, Agilent Technologies) were recorded. System calibration was performed on a BSA standard (Sigma–Aldrich), first resuspended, and then buffer-exchanged into the SEC-MALS running buffer. SEC-MALS data were analyzed with ASTRA version 6.1 software (Wyatt Technology).
6
SEC-MALS Analysis of Protein Samples
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Samples analyzed by SEC-MALS were buffer exchanged by dialysis into SEC-MALS running buffer containing 20 mM HEPES (pH 7.6), 200 mM NaCl, 5% glycerol, 0.5mM TCEP, and 0.05% azide. Samples were applied to a Superose6 10/300 GL (Sigma-Aldrich) column and in-line measurements were recorded for ultraviolet absorbance at 280 nm (using Agilent 1100 series UV detector, Agilent Technologies), static light scattering (DAWN HELEOS 8+light scattering detector, Wyatt Technology), and differential refractive index (Agilent 1200 series refractive index detector, Agilent Technologies) were recorded. System calibration was performed on a BSA standard (Sigma-Aldrich), first resuspended, then buffer exchanged into the SEC-MALS running buffer. SEC-MALS data was analyzed with ASTRA v6.1 software (Wyatt Technology).
7
Western Blotting under Reduced Conditions
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Western blotting was performed under reduced denaturing conditions according to previously described procedures (Li et al., 2014 (link)). Briefly, cells were washed with ice-cold PBS, and soluble proteins were extracted with cell lysis buffer (100 mM Tris-HCl pH = 8, 150 mM NaCl, 1% NP-40, phosphatase and protease inhibitor cocktail tablets (Abcam) according to the manufacturer's protocol). The protein concentration was determined using the Bio-Rad Bradford assay (Bio-Rad, Hercules, CA) and BSA standards (Sigma Aldrich). An equal amount of protein was separated by SDS-PAGE. Antibodies against Src (EMD Millipore) and actin (Sigma Aldrich) were used.
8
Quantitative Alkaline Phosphatase Assay
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For alkaline phosphatase (ALP) staining, cells were soaked in BCIP/NBT solution (Beyotime, China) in darkness, and purple-stained areas were considered positive. For level of ALP activity, the detection was performed following the manufacturer’s instructions (Beyotime, China). Briefly, 400-μl lysis buffer was added to the samples followed by a 4-h incubation at 37°C and a 30-min vibration at room temperature. After that, samples were mixed with p-Nitrophenyl phosphate (p-NPP) and substrate buffer, followed by a 10-min vibration and a 15-min incubation at 37°C. The enzyme activity was measured at 405 nm (BioTek, USA). The protein amount was obtained with the Bradford method in aliquots with the protein assay kit (Bio-Rad, USA), and absorbance was measured at 630 nm with protein concentration was being calculated according to a series of BSA standards (Sigma, USA). ALP activity was determined as absorbance at 405 nm per mg of total cellular proteins.
9
MALDI-TOF Analysis of Cathepsin L Inhibition
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Experiments were performed in two
different buffers, one at pH 4.0 (100 mM sodium acetate, pH 4.0, 50
mM NaCl, 5 mM DTT) and the other at pH 6.0 (100 mM sodium phosphate,
pH 6.0, 50 mM NaCl, 5 mM DTT). Prior to the assay, CatL was activated
in each assay buffer for 25 min at 37 °C and then incubated with
CAA0225 at a molar ratio of 1:10 for 15 min at 37 °C. Samples
were prepared for MALDI-TOF analysis by acidification with 2% trifluoroacetic
acid (TFA) followed by the addition of a 2,5-dihydroxyacetophenone
matrix (Bruker Daltonic).
Mass spectrometry was performed on
an UltrafleXtreme III MALDI-TOF/TOF mass spectrometer (Bruker, Billerica,
MA). Samples were prepared on a standard steel target as described
by Wenzel et al.71 (link) The spectra were acquired
in a linear mode with a mass range of 20–50 kDa. The parameters
used were ion source 1, 25.2 kV; ion source 2, 23.15 kV; lens, 8.84
kV; pulsed ion extraction, 380 ns; detector gating was set to 8 kDa.
The spectra were externally calibrated with aldolase and BSA standards
(Sigma-Aldrich). Acquisition, processing, and calibration were performed
using FlexControl 3.0 and FlexAnalysis software (Bruker).
different buffers, one at pH 4.0 (100 mM sodium acetate, pH 4.0, 50
mM NaCl, 5 mM DTT) and the other at pH 6.0 (100 mM sodium phosphate,
pH 6.0, 50 mM NaCl, 5 mM DTT). Prior to the assay, CatL was activated
in each assay buffer for 25 min at 37 °C and then incubated with
CAA0225 at a molar ratio of 1:10 for 15 min at 37 °C. Samples
were prepared for MALDI-TOF analysis by acidification with 2% trifluoroacetic
acid (TFA) followed by the addition of a 2,5-dihydroxyacetophenone
matrix (Bruker Daltonic).
Mass spectrometry was performed on
an UltrafleXtreme III MALDI-TOF/TOF mass spectrometer (Bruker, Billerica,
MA). Samples were prepared on a standard steel target as described
by Wenzel et al.71 (link) The spectra were acquired
in a linear mode with a mass range of 20–50 kDa. The parameters
used were ion source 1, 25.2 kV; ion source 2, 23.15 kV; lens, 8.84
kV; pulsed ion extraction, 380 ns; detector gating was set to 8 kDa.
The spectra were externally calibrated with aldolase and BSA standards
(Sigma-Aldrich). Acquisition, processing, and calibration were performed
using FlexControl 3.0 and FlexAnalysis software (Bruker).
10
Evaluating CREBH and CHOP Levels in Liver Tissues
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Liver tissues were collected from Bhmt-WT and Bhmt-KO to evaluate CREBH and CHOP levels. Protein extracts were prepared using RIPA lysis buffer (Sigma, ST. Louis, USA) supplemented with protease inhibitor cocktail (Complete, Roche) and sonicated 50 oscillation/sec for 5 min (Tissue Lyser LT, Qiagen). Total protein concentrations for all samples were quantified using BSA standards (catalog number A7030, Sigma-Aldrich). Proteins were loaded into SDS-PAGE gels and blotted on nitrocellulose. Membranes were immersed in blocking solution (BSA 5%; catalog number 03116956001, Sigma; TBS 1x and 0.1% Tween-20) for 1 h. CREBH antibody (catalog number: EWS10. Kerafast; Boston, MA, USA.) was used at 1:1000 dilution, overnight incubation. CHOP (catalog number 5554. Cell Signaling Technology, US.) was used 1:1000, overnight incubation. Beta actin (catalog number ab8226. Abcam, Boston, MA, USA.) was used 1:1000 for 1 h incubation at room temperature. The secondary antibodies were IRDye 800CW goat anti-rabbit IgG (catalog number 926-322. Li-Cor Biosciences, Lincoln, NE, US) and goat anti-mouse IgG IRDye 680RD (926-68070. Li-Cor Biosciences). The membranes were imaged in a Li-Cor Odyssey imaging system and protein abundance was quantified using the Image Studio Lite (Li-Cor Biosciences, version 5.2.5, 2015). Data are presented by mean ± SEM.
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