Tissue and cellular proteins were extracted and quantified. A 10% polyacrylamide gel was used to resolve the proteins. Proteins were transferred onto 0.2 µm PVDF membranes (Merck Millipore, Germany). GAPDH levels were used to normalize loading. A solution of 10% fat-free milk in TBST was used to block the membrane for 2 h at room temperature after which the membranes were rinsed. The proteins were probed using antibodies at 4°C overnight. These antibodies included rabbit anti-human GIT1 (Abcam, Cat. No. ab171956); rabbit anti-p-ERK (Cat. No. #4370), anti-ERK (Cat. No. #4695), anti-E-cadherin (Cat. No. #3195), anti-p-AKT (Cat. No. #4685), anti-Vimentin (Cat. No. #5741), anti-β-cadherin (Cat. No. #8480), anti-AKT (Cat. No. #9272), and anti-GAPDH (Cat. No. #5174), all bought from Cell signaling, and diluted to 1:1000. Membranes were then incubated with HRP-conjugated secondary antibody (Cell signaling, Cat. No. #7074) at 1:2000 for 2 h at room temperature. Chemiluminescence was developed using enhanced ECL chemiluminescent substrate (New Cell & Molecular Biotech, Suzhou, China) and membranes imaged on Chemidoc XRS Gel Imaging System (Biorad). Images were analyzed using ImageJ software.
0.2 m pvdf membrane
Manufactured by Merck Group
Sourced in United States
The 0.2 µm PVDF membranes are a type of laboratory filtration equipment used for the separation and purification of various samples. These membranes have a pore size of 0.2 micrometers and are made from polyvinylidene fluoride (PVDF), a durable and chemically resistant material. The primary function of these membranes is to filter and retain particles, molecules, or organisms larger than the specified pore size, while allowing smaller components to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc imaging system, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Fluorescent secondary antibody, by Cell Signaling Technology (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Murf1, by Abcam (1 mentions)
Mafbx, by Abcam (1 mentions)
Smp30, by Abcam (1 mentions)
Hdac2, by Cell Signaling Technology (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Et1601 4, by Huabio (1 mentions)
Sc 13595, by Santa Cruz Biotechnology (1 mentions)
Sc 13520, by Santa Cruz Biotechnology (1 mentions)
Ecl reagent supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti gfpt2, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
8 protocols using 0.2 m pvdf membrane
1
Quantitative Protein Analysis by Western Blot
2
Western Blot Analysis of CYP3A4, POR, and CB5
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A protocol described by Casabar et al. (2010) was used with modifications for Western blot analysis. Cell lysate total protein was quantified using nanodrop A 280 prior to separation by SDS-PAGE for 120 minutes at 120 V and transferred onto 0.2 µm PVDF membranes (Merck, Kenilworth, New Jersey) using wet transfer technique. The blots were blocked for 1 hour in Trisbuffered saline (TBS) containing 0.01% Tween 20 and 5% BSA at RT. The blots were then incubated with primary antibodies (1:1,000 dilution) of CYP3A4, POR, CB5, or β-actin (loading control) for 1 hour at room temperature. Then, the blots were washed with TBS containing 0.01% Tween 20 before incubation for 1 hour with horseradish-peroxidase-conjugated secondary antibody (1:10,000 dilutions). After further washing with TBS containing 0.01% Tween 20, blots were incubated in chemiluminescence reagents (Azure Biosystems, Dublin, CA). Finally, Molecular Imaging System (Amersham, Kingsport, TN) was used to detect band intensities.
3
Western Blot Analysis of Muscle Proteins
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C2C12 cells and gastrocnemius muscle were lysed with RIPA buffer (Solarbio, Beijing, China); the protein concentrations were measured by a BCA kit (Beyotime, Shanghai, China). Then, 40 µg of the samples were electrophoresed in 10–12% SDS polyacrylamide gels and electrically transferred to 0.2-µm PVDF membranes (Millipore, USA). The membranes were immersed and incubated in 5% fat-free milk at room temperature for 1 hour. The membranes were then incubated with primary antibodies against HDAC2, P53, P21, IKK, and NF-κBp65 (1:1000, Cell Signaling Technology, USA) and MURF1, MAFbx, and SMP30 (1:1000, Abcam, UK) overnight at 4°C, followed by incubation with fluorescent secondary antibodies (1:1000, Cell Signaling Technology, USA). The GAPDH antibody (1:1000, Cell Signaling Technology, USA) was used as the control. Fluorescence signal detection was performed with an infrared imaging instrument (Odyssey, USA), and protein expression was quantified by densitometry analysis with ImageJ software.
4
Protein Expression Analysis in Aortic Tissue
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Abdominal aortas were homogenized and lysed. The concentration of soluble proteins in cell lysates was determined using the BCA assay. Total protein (30 µg) of each sample was separated by SDS-PAGE, transferred to 0.2 µm PVDF membranes (Millipore, Burlington, MA, USA) and probed with primary antibodies against α-SMA (YM3365; Immunoway Biotechnology, Plano, TX, USA), MMP2 (SC-13595; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (sc-13520; Santa Cruz Biotechnology), and GAPDH ( ET1601-4; HUABIO, Woburn, MA, USA). The ECL system was used for detection. GAPDH was used as an internal control, and the results are expressed as the mean ± standard deviation.
5
PBMC Lysis and Immunoblot Analysis
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Peripheral blood mononuclear cells were lysed using 100 µl of Laemmli buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris–HCl). The lysates were transferred onto a 0.2 µm PVDF membrane (Millipore) and probed with anti-IL-1β, anti-ASC, and anti-β-actin antibodies. The blots were developed using a Bio-Rad ChemiDoc Imaging system (Bio-Rad).
6
Western Blot Analysis of GFPT2 Protein
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Tissues were cut and digested by RIPA buffer (Beyotime Biotechnology, Shanghai, China) and the concentration of proteins were measured using BCA™ Protein assay kit (ThermoFisher Scientific). Total 30 μg protein was separated by SDS PAGE gels and transferred to 0.2 µm PVDF membrane (Millipore). After blocking with 5% fat free milk, membrane was incubated with primary antibodies (anti-GFPT2, Thermo Fisher Scientific, PA5-26290, 1:1,000 dilution; anti-GAPDH, abcam, ab8245, 1:5,000 dilution) at 4°C overnight. The next day, the membrane was incubated with secondary antibodies (Cell Signaling Technology, #14708 or #14709, 1:10,000 dilution) for 2 hrs at room temperature. Then the membrane was exposed after using ECL reagent Supersignal West Pico chemiluminescent Substrate (ThermoFisher Scientific).
7
Western Blot Protein Detection Protocol
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Western blot was performed as described previously69 (link) with minor modifications. Briefly, protein samples were heated at 95 °C for 5 min with 2xSDS loading buffer (Invitrogen) and resolved on 12% SDS-PAGE (Invitrogen). Following electrophoresis, the proteins were transferred to PVDF membrane 0.2 µm (Millipore). Primary mouse anti-GFP antibodies (dilution WB 1:1000) (#11814460001, Roche) and secondary goat anti-mouse IgG (H + L) conjugated with HRP (dilution WB 1:5000) (Cat. No. G-21040, Life Technologies) were used for immunoblotting. Bound primary antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies and the Super Signal West Dura ECL detection reagent (Life Technologies). The specific protein bands were visualized using the Super Signal West Dura ECL detection reagent (Life Technologies) and imaged using the ChemiDoc Imaging System (Bio-rad, Gladesville, New South Wales, Australia).
8
Quantifying Nuclear SIRT1 Activity
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Acetylated-histone H3, assayed by dot-blot, was used to evaluate the nuclear SIRT1 activity [30] (link), [31] (link). Samples (0.5 µg) were quickly added to a PVDF membrane (0.2 µm, Millipore) pre-wet with ethanol. Membranes were washed 5 minutes in methanol and twice in TBS. Unoccupied binding sites were blocked 1h30 with blocking solution containing 3% non-fat dry milk in TBS. Then, the membranes were incubated with a primary antibody, rabbit anti- acetylated-histone H3 (1∶6 700; 06-599, Upstate), overnight at 4°C in blocking solution. The acetylated-histone H3 specific immunoreactivity was visualized as described for SIRT1 protein western-blot. Results were expressed in arbitrary units (AU).
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