All salts and solvents, unless otherwise stated, were obtained from Fisher Scientific (UK). Sodium phosphate monobasic monohydrate, low viscosity chitosan from shrimp, glycerol phosphate disodium salt hydrate, tris(hydroxymethyl)amino-methane [(HOCH2)3CNH2], albumin from bovine serum (BSA), albumin from chicken egg white (OVA) and QuantiPro™ Bicinchoninic Acid Assay Kit were obtained from Sigma-Aldrich (UK). l (+)-lactic acid 90 % solution in water was obtained from Acros Organics (USA). Dialysis membranes (size 10, MWCO 12–14 kDa) were obtained from Medicell International Ltd (UK). MWCTs and SWCTs were purchased from Cheap Tubes Inc. (USA). The MWCTs used in the study were 8–15 nm in diameter and 10–50 µm in length. The SWCTs used were 1–4 nm in diameter and 5–30 µm in length. Di-potassium hydrogen orthophosphate anhydrous (K2HPO4) was acquired from British Drug Houses (UK) and troclosene sodium dehydrate from Guest Medical (UK). Water used in all experiments was purified water. NOSC and chitosan modified hydroxyapatite (HACS) were prepared as described previously [12 (link), 21 (link)].
Quantipro bicinchoninic acid assay kit
Manufactured by Merck Group
Sourced in United Kingdom
The QuantiPro™ Bicinchoninic Acid Assay Kit is a colorimetric assay used for the quantification of total protein concentration. The kit utilizes the bicinchoninic acid (BCA) method to measure the reduction of copper ions by proteins in an alkaline environment, with the resulting purple-colored reaction being measured spectrophotometrically.
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Lab products found in correlation
Albumin from chicken egg white ova, by Merck Group (1 mentions)
Sodium phosphate monobasic monohydrate, by Merck Group (1 mentions)
Albumin from bovine serum bsa, by Merck Group (1 mentions)
Glycerol phosphate disodium salt hydrate, by Merck Group (1 mentions)
Tris hydroxymethyl aminomethane hoch2 3cnh2, by Merck Group (1 mentions)
Gel imaging system, by Bio-Rad (1 mentions)
Protease inhibitor, by Keygen Biotech (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Plcγ1, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit immunoglobulin g antibody, by Abcam (1 mentions)
Nfatc1, by Abcam (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Peroxidase linked secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Fluorchem fc2, by Bio-Techne (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
4 protocols using quantipro bicinchoninic acid assay kit
1
Synthesis and Characterization of Chitosan-Based Nanocomposites
2
Western Blot Analysis of Immune Signaling
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Total protein was extracted from cells using lysis buffer containing protease inhibitors (KeyGEN BioTECH, Nanjing, China). Protein concentration was measured using a QuantiPro Bicinchoninic Acid Assay kit (Sigma-Aldrich). Proteins were separated by 15% dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. The membranes were cut according to protein size to incubate antibodies (rabbit monoclonal antibodies against GAPDH (1 : 7500 dilution; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antibodies against Btk (1 : 400; Boster, Wuhan, China), rabbit polyclonal antibodies against PLCγ1 (1 : 7500 dilution; Abcam, Cambridge, UK), and rabbit polyclonal antibodies against NFATc-1 (1 : 7500 dilution; Abcam, Cambridge, UK)) at 4°C overnight. Membranes were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (Abcam, MA, USA) for 2 hours after 3 washes. The membranes were added to an enhanced chemiluminescence (ECL) system (Thermo Scientific, Waltham, MA, USA). Protein bands were visualized using a gel imaging system (Bio-Rad, Hercules, CA, USA). Images were analyzed and outputted using Image Lab (Bio-Rad).
3
Western Blot Analysis of EMT Markers
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Cells were washed twice with ice-cold PBS and solubilized in RIPA buffer (Sigma-Aldrich, Saint Louis, MO) on ice and then was quantified using QuantiPro bicinchoninic acid assay kit (Sigma-Aldrich). Proteins were denatured at 100°C with sample buffer for 5 min. Equal amounts of protein (50 μg) separated by electrophoresis in 10% to 12% SDS-PAGE gels according to their molecular weight. Proteins were transferred onto PVDF membranes (Bio-Rad) and blocked for 2 h in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween 20). The membrane was then exposed to the primary antibody overnight at 4°C. Snail, slug, NF-κB, p-NFκB P65 (Ser536), STAT3, p-STAT3, AKT, p-AKT were purchased from Cell Signaling (Beverly, MA); Twist1, Zeb1, Zeb2, vimentin, E-cadherin, N-cadherin, β-action were purchased from Santa-Cruz Biotechnology, all primary antibodies dilution is 1:1000. After washing, the membranes were incubated for 1.5 h at room temperature with peroxidase-linked secondary antibody (Santa-Cruz). Signals were revealed with an electrochemoluminescence (ECL) Western Blotting Analysis System (Pierce) using the FluorChem®FC2 (Alpha Innotech, CA), and then quantified using ImageJ® software.
4
Hydrogel Release Studies: BSA and OVA
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Release studies were performed including bovine serum albumin (BSA, 1 mg/ml) in the hydrogels (5 ml). After the gelation of the samples, prepared in a 15 ml tubes (n = 4), PBS (5 ml) was added. The samples were then incubated at 37 °C and at set time points 2 ml of PBS were sampled and replaced with 2 ml of fresh buffer. Release studies were also performed including ovalbumin (OVA, 1 mg/ml) in the hydrogels. The suspensions were transferred (1 g per sample) into a Flot-A-Lyzer G2 dialysis device (cut off 50 kDa) and PBS (15 ml) was added to them. The samples were subsequently incubated at 37 °C and at set time points the sampled buffer was replaced with fresh buffer. Determination of both BSA and OVA concentrations was carried out with a QuantiPro™ Bicinchoninic Acid Assay Kit (Sigma, UK) and samples prepared according to manufacturer’s instructions, reading the UV absorbance at 570 nm. Unloaded gels were used as a control to eliminate any false response due to chitosan interaction with the assay kit. The two model drugs were tested with slightly different methods due to limitations imposed by their molecular weight.
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