The implants attached with primary osteoblasts were washed with ice-cold PBS and lysed to release the whole proteins by RIPA buffer with 1 mM PMSF. The cell lysates were agitated at 4 °C for 30 min and then centrifuged at 4 °C for 20 min. The protein concentration was determined by the BCA assay. The protein extracts (30 μg per sample) were separated by 8% or 10% Tris-glycine SDS-PAGE and then transferred onto PVDF membranes (Millipore) after mixed with 5× loading buffer. The PVDF membranes were blocked in TBST (Tris Buffer Saline, 0.5% Tween-20) containing 5% BSA for 1 h and incubated overnight at 4 °C with primary antibodies to OCN (1:1000, Abcam, Cambridge, MA, USA), Runx2 (1:1000, Biorbyt Ltd., Cambridge, UK), Wnt3a (1:1000, Novus Biologicals, Littleton, CO, USA), Lrp6 (1:1000, Lifespan Bioscience, Seattle, WA, USA), β-catenin (1:1000, Millipore, Billerica, MA, USA), β-Tubulin (1:3000, Bioworld technology, Inc., Louis Park, MN, USA), and β-Actin (1:3000, Bioworld) in TBST containing 5% BSA. The membranes were then incubated with a 1:3000 dilution of HRP-conjugated secondary antibody for 1 h at RT, and visualized by an ECL chemiluminescence system (GE ImageQuant 350, GE Healthcare). Semi-quantitative analysis was performed using the QuantityOne Software (Bio-Rad). β-Actin or β-Tubulin was used as an internal control for normalization.
Ge imagequant 350
Manufactured by GE Healthcare
Sourced in United States
The GE ImageQuant 350 is a laboratory imaging system designed for the analysis and quantification of biomolecules, such as proteins and nucleic acids, in various applications. It utilizes advanced imaging technology to capture high-quality images of gels, blots, and other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
β tubulin, by Bioworld Technology (2 mentions)
β catenin, by Merck Group (2 mentions)
Runx2, by Biorbyt (2 mentions)
Wnt3a, by Novus Biologicals (2 mentions)
Pvdf membrane, by Abcam (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
P gsk 3β, by Abcam (1 mentions)
Supersignal west pico chemiluminescent substrate kit, by Thermo Fisher Scientific (1 mentions)
Nc membrane, by Merck Group (1 mentions)
Hif 1α, by Bioworld Technology (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Bca protein assay kit, by Dingguo (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dnase ι, by Takara Bio (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
5 protocols using ge imagequant 350
1
Evaluating Osteoblast Protein Expression
2
Western Blot Protein Extraction Protocol
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The cells were washed with ice-cold PBS and lysed to release the whole proteins using RIPA buffer with 1 mM PMSF. The cell lysates were transferred into a pre-cooled microcentrifuge tube and constant agitation was maintained for 30 min at 4°C. The protein extracts were then centrifuged at 4°C for 20 min at 12,000 rpm. The protein content of the supernatant was collected and the protein concentration was determined by BCA assay (Pierce Chemical Co., Rockford, IL, USA). The protein extracts (30 µg/sample) were subjected to electrophoretic separation by 10% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto PVDF membranes (Millipore), after being mixed with 5X loading buffer and boiled for 8 min. The PVDF membranes were blocked in Tris-buffered saline with 0.5% Tween-20 (TBST) containing 5% BSA for 2 h, and incubated overnight at 4°C with primary antibodies to His-probe (1:500), GAPDH (1:1,000) and Runx2 (1:400) in TBST containing 5% BSA. The membranes were then incubated with a 1:5,000 dilution of HRP-conjugated goat anti-rabbit secondary antibody for 1 h at room temperature, and then visualized using an ECL system (GE ImageQuant 350; GE Healthcare, Piscataway, NJ, USA). GAPDH was used as an internal control for normalization. Semi-quantitative analyses of the bands were performed by using the Quantity One software (Bio-Rad).
3
Protein Extraction and Western Blotting for Analyzing Cellular Responses to PEMF
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The procedures of protein extraction and western blotting were described as in details in our previous study29 (link). In brief, cells in the PEMF group were exposed to PEMF at 37 °C for 3 days (2 h/day), and then protein lysates were prepared were prepared using RIPA buffer supplemented with 1 mM PMSF on ice. After quantification, protein extracts in equal amounts were loaded on a 10% Tris–glycine SDS-PAGE gel, and then electrotransferred to PVDF membranes. The blots were blocked in 5% bovine serum albumin (BSA) for 1 h, followed by incubation with primary antibodies against OCN (Abcam, Cambridge, MA), Runx2 (Biorbyt Ltd., Cambridge, UK), Wnt3a (Novus Biologicals, Littleton, CO), p-GSK-3β (Abcam), β-catenin (EMD Millipore, Billerica, MA) and β-tubulin (Bioworld technology, Inc., Louis Park, MN) in TBST containing 5% BSA overnight at 4 °C. β-tubulin was employed as the protein loading control. After incubation with a 1:3000 dilution of HRP-conjugated secondary antibody for 1 h at room temperature, the membranes were visualized using the ECL chemiluminescence system (GE ImageQuant 350, GE Healthcare). The Quantity One Software (Bio-Rad) was used to quantify the densities of the bands.
4
Cardiac Protein Extraction and Western Blot
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Total protein was extracted from cardiac muscle tissues using RIPA buffer supplemented with PMSF. The protein concentration was quantified using a BCA protein assay kit (DingguoChangsheng Biotech Co., Beijing, China). The protein was separated in 6% or 10% SDS-PAGE and transferred to the NC membrane (Millipore Corp., Bedford, Mass, United States) by electroblotting. Then, membranes were blocked in 5% skim milk for 1 h at room temperature and incubated at 4°C overnight with the primary antibodies of HIF-1α, ACE, ACE2, and GAPDH (Bioworld, Atlanta, GA, United States). After washing with Tris-buffered saline containing 0.5% Tween-20 (TBST), membranes were incubated with the secondary antibody for 2 h at room temperature. The blots were developed using the SuperSignal West Pico chemiluminescent substrate kit (Thermo Scientific, Rockford, IL, United States) and visualized by an ECL chemiluminescent system (GE ImageQuant 350, GE Healthcare).
5
Transcriptome Analysis of Trichophyton mentagrophytes
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The T. mentagrophytes wild-type strain ATCC 28185 (a gift from Ruoyu Li, Peking University First Hospital, China) was maintained at 28 °C on solid Sabouraud dextrose medium (SDA) for 14 days. Five mL of sterile saline was used to wash off spores so as to collect fungus liquid. The fungus liquid concentration was adjusted to 108 CFU/mL by cell count plate. SDA with 1 mM EDTA was supplemented to generate zinc deficient SDA, named SDA-Zn (zinc ions have been chelated). The 150-μL fungus liquid was inoculated to SDA (sufficient zinc ions, grouped into Norm) and SDA-Zn with 200, 400, 600, and 1000 μM of zinc sulfate (grouped into Zn200, Zn400, Zn600, and Zn1000) respectively. Culture conditions were 28 °C for 14 days.
Total RNA was extracted using TRIzol® reagent (Invitrogen, USA) following the manufacturer’s protocol, and DNase Ι (Takara, Japan) was used to remove genomic DNA. Integrity and size distributions were checked using an Agilent 2100 (Agilent, USA) with an RNA integrity number (RIN: 8.0) and GE Image Quant 350 (GE Healthcare , USA).
Total RNA was extracted using TRIzol® reagent (Invitrogen, USA) following the manufacturer’s protocol, and DNase Ι (Takara, Japan) was used to remove genomic DNA. Integrity and size distributions were checked using an Agilent 2100 (Agilent, USA) with an RNA integrity number (RIN: 8.0) and GE Image Quant 350 (
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