Vero cells or SK-N-SH cells were infected with HSV-1 (MOI = 1) in the presence or absence of amentoflavone. Cell lysates were collected at various time points using SDS buffer (Beyotime, ShangHai, China), and proteins were separated by 10% gradient SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane (Millipore), and were then blocked with 5% nonfat milk for 1 h at room temperature. Targeted proteins were incubated with primary antibodies overnight at 4 °C and with the secondary antibodies for 1 h at room temperature. Finally, those target proteins were detected by ECL solutions. The band intensity of each protein was calculated using Quantify One software (Bio-Rad, Hercules, CA, USA) and was normalized to that of GAPDH.
Quantify one software
Manufactured by Bio-Rad
Sourced in United States
Quantify One software is a data analysis tool designed for life science researchers. It provides functionality for quantifying and analyzing data from various experiments, including Western blots and gel-based assays. The software allows users to import, process, and visualize experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phosphorimager, by Bio-Rad (1 mentions)
Random primer labeling kit, by Agilent Technologies (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Sds page buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Loading buffer, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Luminata crescendo western hrp substrate, by Merck Group (1 mentions)
Prism software, by GraphPad (1 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
8 protocols using quantify one software
1
Amentoflavone Inhibits HSV-1 Infection
2
Genomic DNA Analysis of Recombination
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Genomic DNA preparation and physical analysis of recombination were performed as described previously (19 (link),35 (link)–36 (link)). For 2D gel electrophoresis, genomic DNA was digested with 80 units of XhoI enzyme, and loaded onto 0.4% SeaKem Gold agarose gel in Tris-Borate-EDTA (TBE) buffer, at ∼1 V/cm for 21 h. Gels were stained with 0.5 μg/ml ethidium bromide (EtBr) for 30 min, and afterward the lanes containing DNA of interest were cut out to prepare gel slices. Following this, SeaKem LE agarose (0.8%) with 0.5 μg/ml EtBr was added to the 2D gel tray. The second gel electrophoresis was carried out at ∼6 V/cm, for 6 h at 4°C. Southern blot analysis was performed using 32P-dCTP–labeled radioactive nucleotides, prepared with the Random Primer labeling kit (Agilent Technologies, USA). DNA hybridization signals were quantified using a Bio-Rad Phosphorimager and Quantify One software (Bio-Rad, USA). Detection of DSBs in the rad50S background was performed as described previously (19 (link)).
3
Protein Extraction and Western Blot
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Cell samples were rinsed thrice with precooled PBS and lysed in SDS buffer (Beyotime) containing 1 mM PMSF (Beyotime). Lysates were heated at 100°C for 15 min. Insoluble cell debris was discarded following centrifugation at 12,000 × g for 10 min at 4°C. The protein concentrations of retained supernatant was measured using an enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime). Subsequently, the lysates were mixed with calculated volumes of 5× SDS-PAGE buffer (Beyotime) and SDS buffer to obtain equivalent protein concentrations and then boiled for 10 min. Finally, samples were separated using 8-10% gradient SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany), and probed with indicated primary antibodies and then with HRP-conjugated secondary antibodies (Invitrogen). Immunoreactive bands were detected using enhanced chemiluminescence (Millipore), and the band intensity of each target protein was calculated using Quantify One software (Bio-Rad, Hercules, CA, USA) and normalized to GAPDH band intensity.
4
PrP Glycoform Profile Analysis
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Brains were homogenized in 0.32 M sucrose in PBS. Total protein concentration was determined using the bicinchoninic acid assay (Pierce) according to manufacturer’s instructions. Samples were adjusted to 1 μg/μl and digested with proteinase K (PK) (20 μg/μl) in PBS, 0.5% SDS and 0.5% NP-40 for 30 minutes at 37 °C. Proteinase K reaction was stopped by adding loading buffer (Invitrogen) followed by boiling samples for 5 minutes at 95 °C. PK-treated and untreated samples were separated on a 12% Bis-Tris polyacrylamide gel (NuPAGE, Invitrogen) and blotted onto a nitrocellulose membrane. Anti-PrP antibody (POM1, 200 ng ml−1) [16 (link)] was used as a primary antibody which was detected using rabbit anti-mouse IgG1 conjugated to horseradish peroxidase (HRP). Western blots were developed using Luminata Crescendo Western HRP substrate (Millipore) and visualized using the FUJI-FILM LAS-3000 system. The glycoform profiles of PrPSc after PK treatment were quantified using the Quantify One software (BioRad). The relative intensity of each PrPSc glycoform (i.e. di-, mono-, ungylcosylated) was measured which was expressed then as a percentage of the total signal. Statistical analysis (two-way ANOVA) was performed using the Prism software (www.Graphpad.com ).
5
VCAM-1 Detection by SDS-PAGE and Western Blot
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Membrane protein was analysed by SDS-PAGE. Briefly, 15 µL samples were homogenised in SDS-PAGE sample loading buffer and electrophoresed. Protein bands were stained using Coomassie brilliant blue. In addition, Western blot analysis was applied for further identification of VCAM-1. Proteins were transferred to nitrocellulose membranes. The membranes were blocked with blocking buffer for 1 h at room temperature and incubated with anti-bovine VCAM-1 polyclonal antibody (1:2,000 dilution). After five washes with PBS-T, the membranes were incubated with goat anti-rat IgG (1:20,000 dilution). Proteins were visualised by incubation with enhanced chemiluminescence detection system reagents (Amersham Pharmacia Biotech, USA) and the amount of target protein was calculated by gray scanning with Quantify One software (Bio-Rad). The level of VCAM-1 protein expression was expressed as the ratio of VCAM-1 to β-actin expression.
6
Quantifying RhoA and Nogo-A Protein Levels
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After Trizol extraction, the remaining fraction was centrifuged at 1,500 r/min for 30 minutes. The supernatant was used for total protein concentration determination using the Bradford method (Yang et al., 2010). The protein samples were electrophoresed on a 10% SDS-PAGE resolving gel and transferred onto a polyvinylidene difluoride membrane at 14 V for 14 hours. Then, the membrane was blocked for 2 hours at 37°C, washed in TBS three times for 10 minutes each, and treated with rabbit anti-rat RhoA monoclonal antibody (1:800; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-rat β-actin monoclonal antibody (1: 500; Santa Cruz Biotechnology) at 4°C overnight. Thereafter, the membrane was washed in TBST four times for 5 minutes each and incubated with goat anti-rabbit IgG (1:700; Santa Cruz Biotechnology) at 37°C for 1.5 hours, washed in TBST four times for 5 minutes each, in TBS for 10 minutes, and developed with 3,3′-diaminobenzidine (DAB). Optical density ratios of RhoA and Nogo-A to β-actin were determined using Quantify One software (Bio-Rad, Hercules, CA, USA) to calculate the respective protein expression levels.
7
Protein Extraction and Western Blot Analysis
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Cells were lysed in sodium dodecyl sulfate (SDS) buffer (Beyotime) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (Beyotime), and the protein concentration was measured using an enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime). The cell lysates were then mixed with the appropriate volume of 5× SDS-polyacrylamide gel electrophoresis (PAGE) buffer (Beyotime) and SDS buffer to obtain the same concentration and then boiled for 10 min. Finally, samples were analyzed by SDS-PAGE on 8–15% gradient gels, transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany), probed with the indicated primary antibodies, and then incubated with horseradish peroxidase-conjugated secondary antibodies. All proteins of interest were detected by enhanced chemiluminescence (Millipore). The band intensity of each protein was calculated using Quantify One software (Bio-Rad, Hercules, CA, USA) and normalized to that of GAPDH.
8
Protein Expression Analysis of BMSCs
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BMSCs and hematopoietic cells were collected respectively, and fty microgram of protein samples were loaded for SDS-PAGE and transferred onto 0.2µm PVDF membrane, which were incubated with the primary antibodies against p53, p21, p16, p-p38. β-actin was used as the internal control protein. After appropriate HRP-conjugated secondary antibody incubation in TBST, protein bands were detected using an ECL detection system. The semi-quanti caton analysis was performed using Quantify One software (BioRad, Hercules, CA, USA).
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