The tissue surrounding the implant was frozen in liquid nitrogen and then ground with a chilled mortar and pestle. RIPA buffer containing 1 mM PMSF (KeyGEN Biotech, China) was used to lyse the tissue and release whole protein. Protein concentration was measured by using a BCA assay kit (KeyGEN Biotech). Next, 30 μg of protein mixed with 5X loading buffer was separated by 12% Tris-glycine SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were incubated in TBST (Tris-buffered saline, 0.5% Tween-20) containing 5% nonfat dry milk for 1 h at room temperature and incubated with different primary antibodies (OCN, 1:1000, Abcam, UK; Runx2, 1:1000, Abcam; OPG, 1:1000, Abcam; RANKL 1:1000, Abcam; β-catenin, 1:1000, Abcam; SOST, 1:1000, Abcam; β-actin, 1:1000, Cell Signaling Technology, Danvers, USA; GAPDH, Cell Signaling Technology, USA) overnight at 4°C. The membranes were washed with TBST 3 times and incubated with the secondary antibodies (1:5000, ZSGB-BIO, China) for 1 h at room temperature. ECL western-blotting detection reagent (KeyGEN Biotech) was used to test the bands. Quantity One Software (Bio-Rad, Hercules, USA) was used for semi-quantitative analysis.
Bca assay kit
Manufactured by Keygen Biotech
Sourced in China
The BCA assay kit is a colorimetric assay used for the quantitative determination of total protein concentration. It utilizes the bicinchoninic acid (BCA) reaction to measure the reduction of copper ions by proteins in an alkaline environment.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (13 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Total protein extraction kit, by Keygen Biotech (3 mentions)
Ripa buffer, by Keygen Biotech (3 mentions)
Chemiluminescence detection system, by Bio-Rad (2 mentions)
Whole cell lysis assay, by Keygen Biotech (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Protein extraction reagent, by Keygen Biotech (2 mentions)
Gapdh, by Proteintech (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Runx2, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Keygen Biotech (1 mentions)
β catenin, by Abcam (1 mentions)
50 protocols using bca assay kit
1
Western Blot Analysis of Bone Markers
2
Protein Extraction and Western Blot Analysis
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After extensively washing, cells were lysed using Whole Cell Lysis Assay and proteins were harvested according to the protocol (KeyGen Biotech Inc., Nanjing, China). Protein concentrations was determined by the BCA Assay kit ((KeyGen Biotech Inc., Nanjing, China). Total protein of each lysate was then separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, MA, USA). Afterwards, the membranes were blocked with 5% non-fat milk in TBST at room temperature for 1 h, followed by incubated with antibodies against TLR4 (1:2,000, cat. no. 14358, Cell Signaling Technology, Inc), or IRAK4 (1:2000, cat. no. 4363, cell Signaling Technoloy, Inc), or GAPDH (1:2,000, ab8245, Abcam) overnight at 4 ℃. After washing with TBST, membranes were striped with appropriate HRP-conjugated secondary antibodies for 1h at room temperature, followed by visualized using the Luminol reagent (Thermo Fisher Scienti c, Carlsbad, CA, USA). Eventually, bands were imaged and densitometric analyzed by a chemiluminescence detection system (Bio-Rad, USA).
3
Cellular Uptake of PRN Nanoparticles
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HCEpiC were seeded in a 24-well plate (5×104 cells/well) and incubated for 6 days at 37°C. Subsequently, cells were exposed to PRN, CG-VV-PRN (3.8×10−4% or 0.008% [w/v] CG-VV), PRN-LDH nanoparticles, CG-VV-PRN-LDH nanoparticles or CG-VV-PRN-LDH nanosheets (2 μg/mL PRN) for 1, 2 and 4 h at 37°C, respectively. Once the uptake was finished, the cells were rinsed twice with cold DPBS, trypsinized and resuspended in an appropriate volume of cold DPBS. Intracellular PRN concentration was measured by HPLC. Total protein concentration of cell samples was determined using the BCA assay kit (Cat No KGP905; KeyGen BioTech). The concentration of protein was calculated and calibrated with a standard curve (y =0.001x +0.139, R2=0.9977, where y is the absorption measured at a wavelength of 570 nm using a microplate reader, x is the concentration of standard bovine serum albumin solution ranging from 25 to 500 μg/mL). The uptake index (UI) was calculated using the following formula of UI = C/P, where C is the concentration of PRN and P is the concentration of protein in cell samples.
4
Western Blot Analysis of Extracellular Vesicle Proteins
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Whole‐cell and sEV lysates were prepared by adding RIPA lysis buffer containing 1 mm PMSF, and protein concentration was measured using a BCA Assay Kit (KeyGen Biotech, Nanjing, China). 10–20 µg extracted proteins were separated on gradient SDS/PAGE gels and then transferred to PVDF membranes. The membranes were subsequently incubated at 4 °C overnight with the primary antibodies, including Calexin, Alix, TSG101, CD63, TGF‐β1, GAPDH, Smad2, phopho‐Smad2, E‐cadherin, Snail, Twist 1, and β‐actin. Thereafter, the membranes were incubated at room temperature for 2 h with HRP‐conjugated secondary antibody (Santa Cruz Biotechnology). All blots were detected using the enhanced chemiluminescence (ECL) with ChemiDoc™ XRS+ imaging system (Bio‐Rad, Irvine, CA, USA). quantity one software (Bio‐Rad) was applied to evaluate the image of blots and further normalized by GAPDH and β‐actin.
5
Western Blot Analysis of EMT and Stemness Markers
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In brief, cells were lysed with RIPA buffer containing 1% protease inhibitor cocktail (Roche, Germany) on ice for 30 min and then lysates were centrifuged. Protein concentrations were measured using the BCA assay kit (KeyGen). Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies used in western blot were: anti-HES1 (1:500, ab71559, Abcam), anti-Twist (1:1000, 46702, CST), anti-Snail (1:1000, 3829, CST), anti-Slug (1:1000, 9585, CST), anti-ZEB1 (1:1000, 3396 , CST), anti-ZEB2 (1:1000, 97885, CST), anti-SOX2 (1:1000, ab92494, Abcam), anti-OCT4 (1:1000, 2750, CST), anti-Nanog (1:1000, ab109250, Abcam) and anti-β-actin (1:1000, ab8227, Abcam). All western blots were derived from the same experiment and were processed in parallel.
6
Isolation and Characterization of sEV
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The isolation of the sEV protocol has been described in detail.18 (link) Total exosome isolation reagent (Life, USA) was added to the concentrated solution, put into a 4 °C refrigerator overnight and centrifuged at 10,000 g for 1 h, and the sEV precipitate was stored in a −80 °C refrigerator for later use. Two types of sEV were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Each batch of sEV preparations was ascertained to have a similar size and markers (CD63, Hsp70, and Tsg101). The protein concentrations of sEV were assessed by a BCA assay kit (KeyGEN Biotech, Nanjing, China) following the manufacturers’ instructions.
7
Western Blot Analysis of Exosomes
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The samples from RBC-derived exosomes, BSA-PEG-micelles, and the peak fractions for the BSA-PEG-lipid-exosomes from size-exclusion chromatography were analyzed by Western blotting. The protein concentrations of all samples were estimated using the BCA assay kit (KeyGEN Biotech). Then, equal amounts of protein were separated on a 10% SDS-PAGE and transferred to the PVDF membrane (Millipore) using a high quality wet protein transfer system (L00686C eBLOT L1, Genscript, Nanjing, China). The membranes were then blocked with 5% skim milk powder in TBST buffer for 1 h at RT. The blots were then incubated overnight at 4℃ with the primary antibodies including mouse anti-BSA (1:2000, Proteintech), mouse anti-human CD63 (1:1000, Santa Cruz), mouse anti-human CD9 (1:1000, Proteintech), or mouse anti-human Alix (1:1000, Santa Cruz). Subsequently, the blots were incubated with HRP-conjugated goat anti-mouse IgG antibodies (1:5000, Cell signaling) for 1 h at RT. Then, the blots were developed using the Enhanced chemiluminescence detection kit (BL520A, Biosharp).
8
Protein Quantification and Co-Immunoprecipitation in hiPSC-CMs
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BCA assay kit (KeyGen Biotech, China) was used to quantify the concentration of proteins that were extracted from hiPSC-CMs using lysis buffer. Electrophoresis was used to separate protein samples, which were then transferred to PVDF membranes (Millipore Sigma, China). The primary antibodies were incubated overnight at 4 °C after the membranes had been blocked with skim milk. An ECL imaging system (Bio-Rad, USA) was used to visualize the primary antibodies after they had been conjugated with HRP-conjugated IgG secondary antibody.
Co-IP assays were performed according to the manufacturer’s instructions of the Co-IP kit (Absin, China). Primary antibody (2.5 μg) and cell lysates were mixed overnight at 4 °C. The cell lysates were subsequently mixed with Protein A and Protein G at 4 °C for 3 h. The samples were then centrifuged and washed, and the precipitate was retained. Finally, 30 µl of 1× SDS buffer was added and then boiled, and the supernatant was collected for western blotting after centrifugation.
Co-IP assays were performed according to the manufacturer’s instructions of the Co-IP kit (Absin, China). Primary antibody (2.5 μg) and cell lysates were mixed overnight at 4 °C. The cell lysates were subsequently mixed with Protein A and Protein G at 4 °C for 3 h. The samples were then centrifuged and washed, and the precipitate was retained. Finally, 30 µl of 1× SDS buffer was added and then boiled, and the supernatant was collected for western blotting after centrifugation.
9
Protein Expression Analysis in Cells
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Different groups of cells were lysed in RIPA lysis buffer (keyGEN), and total protein concentration was measured by BCA assay kit (KeyGEN). Western blotting was performed as described previously [24 (link)] with rabbit anti-TGFβ1 pAb (dilution 1:500, BOSTER), rabbit anti-HDAC4 mAb (dilution 1:1,000, CST), rabbit anti-vimentin mAb (dilution 1:1,000, CST), rabbit anti- E-Cadherin mAb (dilution 1:1,000, CST), rabbit anti-MMP–2 mAb (dilution 1:1,000, CST) and rabbit anti-MMP–9 mAb (dilution 1:1,000, CST), whereas a rabbit anti-β-actin monoclonal antibody (dilution 1:1,000, CST) detected the internal control protein.
10
Determination of Alkaline Phosphatase Activity
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ALP activity was determined by the ALP assay kit (Abnova) according to the manufacturer’s instructions. On the test day, the cells were disrupted and corrected by total protein content with the BCA assay kit (KeyGEN,). The absorbance was read at 405 nm in a microplate reader (MK3, Thermo).
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