PLGA nanoparticles loaded with UK (PUNPs) were used as the control group. PUNPs were prepared in the same method as PPUNPs. Take equal amounts of PUNPs and PPUNPs for freeze-drying. Then, the lyophilized powder of nanoparticles was added to 2 mL of PBS solution (pH = 7.4). The PUNPs and PPUNPs solution were placed in an ultrasonic cleaner to ultrasound treatment for 5 minutes. After ultrasound treatment, the NPs were performed to centrifugate. Then the released UK in the supernatant were measured by using the Bradford protein concentration assay kit (Biyuntian, China).
Bradford protein concentration assay kit
Manufactured by Beyotime
Sourced in China
The Bradford protein concentration assay kit is a colorimetric assay used to determine the concentration of protein in a sample. It utilizes the principle of protein-dye binding, where the dye Coomassie Brilliant Blue G-250 binds to proteins, resulting in a color change that can be measured spectrophotometrically. The kit provides the necessary reagents and instructions to perform the assay.
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Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Dichloromethane, by Chron Chemicals (1 mentions)
Paraformaldehyde fix solution, by Beyotime (1 mentions)
Native tissue cell lysate kit, by Solarbio (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dapi staining solution, by Beyotime (1 mentions)
Triton x 114, by Merck Group (1 mentions)
Toxinsensor chromogenic lal endotoxin assay kit, by GenScript (1 mentions)
Peg 6000, by Biosharp (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sodium taurocholate, by Merck Group (1 mentions)
A phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Odyssey software, by LI COR (1 mentions)
Edta free protease inhibitor tablet, by Roche (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
14 protocols using bradford protein concentration assay kit
1
PLGA Nanoparticles for UK Release
2
Preparation and Characterization of PLGA Nanoparticles
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PLGA (MW: 90000, LA: GA = 50:50) was purchased from Jinan Daigang Bioengineering Co., Ltd. (Jinan, China); dichloromethane was purchased from Chengdu Kelong Chemical Co., Ltd. (Chengdu, China); UK and polyvinyl alcohol (PVA-210, MW: ~67,000) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); PFP was purchased from Strem Chemicals, Inc (Massachusetts, USA); Isopropanol was purchased from Chongqing Sichuan East Chemical Co., Ltd. (Chongqing, China); Bradford protein concentration assay kit, DAPI staining solution and 4% paraformaldehyde fix solution was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. (Shanghai, China); DSPE-PEG-FITC, DSPE-PEG-RGD (the MW of PEG is 2000) were purchased from Hunan Huateng Pharmaceutical Co., Ltd. (Hunan, China); CCK-8 kit was purchased from Dojindo Institute of Chemistry (Japan); The native tissue/cell lysate kit was purchased from Beijing Solarbio Science & Technology Co.,Ltd. (Beijing, China); Dulbecco’s modified Eagle’s medium (DMEM), 1640 medium, fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Gibco (USA).
3
Measuring Protein Solubility via HIU Pretreatment
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Protein solubility was measured according to the method reported by Sui, et al. [31] (link) with slight modifications. The 7S solution (15 mg protein/ml 10 mM neutral phosphate buffer) was fully hydrated for 12 h before being pretreated with different HIU pretreatment time (0, 5, 10, 15, and 20 min) and then centrifuged (12,000, 20 min, 4 °C). The protein level of the supernatant was measured using the Bradford Protein Concentration Assay Kit (P0006C, Biyuntian Biotechnology Co., Ltd., Shanghai, China). Protein solubility was expressed as a percentage of supernatant protein concentration to total protein concentration.
4
Recombinant CpStefin Protein Expression and Purification
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The Escherichia coli strain BL21(DE3) harboring recombinant plasmid pGEX-4T-CpStefin preserved in our laboratory was activated and was expanded to an optical density (OD) value of 0.6–1.0 followed by induction using IPTG with a final concentration of 1 mmol. After being shaken at 16 °C at 180 r/min overnight, the cultured product was collected and centrifuged at 10,000 rpm for 15 min followed by ultrasonic disruption on ice (work for 3 s, interval for 4 s, repeat for 20 min). Then, the expression of rCpstefin in the supernatant was determined by SDS-PAGE electrophoresis. Afterwards, rCpstefin was purified by GST•BindTM resin, identified by SDS-PAGE electrophoresis, and the protein concentration was determined by using a Bradford Protein Concentration Assay Kit (Beyotime Biotechnology, Shanghai, China).
The principle of liquid-phase separation was applied to remove endotoxin from rCpstefin using Triton X-114 (Sigma Aldrich, Darmstadt, Germany) according to instruction of manufacture, followed by filtration with a 0.22 μm filter. Afterward, the level of endotoxin in rCpstefin was measured using a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA). The level of endotoxin rCpstefin less than 1 EU/mg (<0.1 ng/mg) was acceptable during cell culture and was applied to treat RAW264.7 cells in the following experiment [31 (link)].
The principle of liquid-phase separation was applied to remove endotoxin from rCpstefin using Triton X-114 (Sigma Aldrich, Darmstadt, Germany) according to instruction of manufacture, followed by filtration with a 0.22 μm filter. Afterward, the level of endotoxin in rCpstefin was measured using a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA). The level of endotoxin rCpstefin less than 1 EU/mg (<0.1 ng/mg) was acceptable during cell culture and was applied to treat RAW264.7 cells in the following experiment [31 (link)].
5
Biochemical Assays of Cellular Enzymes
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Coptisine (purity > 98%) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) kits were purchased from Solarbio (Beijing, China). Alkaline phosphatase (AKP), succinate dehydrogenase (SDH), and lactate dehydrogenase (LDH) activity assay kits were obtained from the Nanjing Jiancheng Bioengineering Institute (Jiancheng, Nanjing, China). The Bradford protein concentration assay kit was purchased from Beyotime Biotechnology (Shanghai, China).
6
Leaf Protein Extraction and Infiltration
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Forty-five-day fresh leaves of trans-N21 tobacco were cut and immediately ground into a powder using liquid nitrogen. The samples were placed into 1 mL of plant protein extraction buffer (50 mM Tris-HCl (pH 7.0), 10 mM MgCl2, 1 mM EDTA, 5 mM DTT, 5% PVP, 10% glycerine) and 10 μL PMSF (100 mM), shocked for 10 min, and incubated for 3 h at 4 °C. The samples were centrifuged at 12,000 r/min for 20 min at 4 °C, and the supernatant was taken. The supernatant was dried then dissolved in 500 μL sterile water, and measured protein concentration via Bradford protein concentration assay kit (Beyotime, P0006) for the detection of protein activity. A small hole was created in the lower epidermis of a fully expanded leaf of Xanthi tobacco, and the prepared protein solution was injected into the hole using a 1-mL needle. The plants were cultured in a 16 h/8 h light/dark cycle at 25 °C for 24 h to observe the results. Proteins of EV tobacco and Xanthi tobacco were extracted as negative controls. Proteins of N21 and Hpa1 directly expressed by E. coli BL21 cells were used as positive controls. The experiment was repeated three times with the same results.
7
Purification of IgY from Egg Yolk
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IgY was purified from the egg yolk mixture when the yolk antibody titer turned relatively steady. The IgY purification protocols, preparing a water-soluble fraction (WSF) of yolk and precipitating IgY in polyethylene glycol (PEG), were proposed by Akita and Polson [29 (link),30 (link)]. Briefly, the egg yolk was solubilized in PBS. The yolk granules were pelleted by centrifugation at 10,000× g for 20 min at 4 °C, the supernatant (WSF) was filtered and added a concentration of 3.5% polyethylene glycol 6000 (PEG 6000, Biosharp, Beijing, China). After being stirred for 30 min at room temperature, the mixture was centrifuged at 10,000× g for 20 min at 4 °C. The supernatant (filtered through three layers of filter paper) was collected and added PEG 6000 to the final polymer concentration of 12% (w/v). The mixture was centrifuged at 10,000× g for 30 min at 4 °C and collected the pellets, resuspended in PBS subsequently. The purity and yield of the IgY were monitored by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the IgY was determined using the Bradford protein concentration Assay kit (Beyotime, Shanghai, China).
8
Western Blot Analysis of TIPE2 Protein
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The PBMCs were lysed with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) and the total protein concentration was determined using a Bradford Protein Concentration Assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. A total of 30 µg protein was separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline containing Tween 20 (TBST) at 4°C overnight, the membranes were incubated with the following primary antibodies at 4°C overnight: Rabbit polyclonal to TIPE2 (dilution, 1:500; cat. no., ab170258; Abcam, Cambridge, MA, USA), rabbit polyclonal to GAPDH (dilution, 1:2,000; cat. no. ab9485; Abcam). GAPDH antibody was used as an internal control. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution, 1:4,000; cat. no. ab6721; Abcam) at room temperature for 2 h. Detection of the signals was performed using an ECL Western Blotting kit (Pierce Biotechnolgy, Inc., Rockford, IL, USA) by exposure to X-ray films (Phenix Research Products, Candler, NC, USA) for 5 min. Protein was quantified using a ScanJet 4C Flatbed Scanner (Hewlett-Packard, Palo Alto, CA, USA) with NIH Image v1.52 software (rsb.info.nih.gov/nih-image/ ).
9
Rat Model of Acute Pancreatitis
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A total of 48 healthy male Sprague Dawley rats (weight, 350±30 g; age, 12–14 weeks), were provided by the Experimental Animal Center of the Third Xiangya Hospital (Changsha, China). Sodium taurocholate was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Amylase polyclonal antibody (cat. no. YT5164) and enzyme-linked immunosorbent assay (ELISA) kit for HMGB1 (Total HMG-1 Cell-Based Colorimetric ELISA kit) were obtained from ImmunoWay Biotechnology Company (Plano, TX, USA). Rabbit anti-HMGB1 (cat. no. ab79823) and anti-Beclin 1 (cat. no. ab55878) primary antibodies were from Abcam (Cambridge, MA, USA). Goat anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA; cat. no. 111-005-144). The color pre-stained protein marker was purchased from Fermentas (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The Bradford protein concentration assay kit was obtained from Beyotime Institute of Biotechnology (Haimen, China). TRIzol® reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The SYBR Green quantitative polymerase chain reaction (qPCR) Mix was purchased from Toyobo Co., Ltd. (Osaka, Japan).
10
Immunoassay Reagent Procurement
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HRP-labeled goat anti-rabbit IgG (SA00001-3, 1:5,000) was purchased from Proteintech Group; the fetal bovine serum (Catalog No: 04-001-1A) was purchased from the Israeli company of Biological Industries; the Bradford protein concentration assay kit (Catalog No: P0013) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and the Mouse IFN- γ (Catalog No: 70-EK280/3-96) and IL-4 (Catalog No: 70-EK204/2-96) ELISA kits were purchased from Multisciences Biotech Co.
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