The cell lysates were prepared using radioimmunoprecipitation assay buffer (RIPA buffer; Sigma-Aldrich, St-Louis, MO, USA) or urea solution (9 M urea, 2% Triton-100, 5% 2-mercaptoethanol), separated on 4–15% gradient gels (Bio-Rad, Richmond, CA, USA) and blotted on polyvinyl difluoride (PVDF) membranes (Bio-Rad). An ECL Prime kit (GE Healthcare, Little Chalfont, UK) was used for signal detection. The antibodies used are listed in S2 Table . ImageJ (NIH) was used for quantification of the signals.
Polyvinyl difluoride pvdf membranes
Manufactured by Bio-Rad
Sourced in United States
Polyvinyl difluoride (PVDF) membranes are a type of laboratory equipment used for protein and nucleic acid transfer and detection in various analytical techniques, such as Western blotting and Northern blotting. They provide a stable and reliable substrate for the immobilization and transfer of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime kit, by GE Healthcare (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ne per nuclear extraction kit, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris gel electrophoresis, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Bca protein quantitative analysis kit, by Beyotime (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Coomassie brilliant blue, by Bio-Rad (1 mentions)
Cxcr1, by Santa Cruz Biotechnology (1 mentions)
Anti cxcr4, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Sartorius (1 mentions)
Anti β actin, by MultiSciences Biotech (1 mentions)
5 protocols using polyvinyl difluoride pvdf membranes
1
Western Blot Protocol for Protein Analysis
2
Sulforaphane and Aesculin Modulate Nrf2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW 264.7 cells (1 × 106 cells/well) were treated with sulforaphane, aesculin, or 3-O-β-d -glycosyl aesculin. Nuclear proteins were isolated by using an NE-PER nuclear extraction kit with the manufacturer’s protocol (Thermo Scientific; IL, USA). The amount of protein was determined by the Bradford assay (Bio-Rad). Equal amounts of protein were separated by 8% NuPAGE® Bis-Tris gel electrophoresis (Invitrogen) in MOPS running buffer and transferred to polyvinyldifluoride (PVDF) membranes (Bio-Rad). Membranes were incubated with 1% bovine serum albumin and then with antibodies overnight at 4 °C (1:1000 dilution for Nrf2 or 1:5000 for HA). The membranes were stripped by a western blot stripping solution per the protocol by the manufacturer (Thermo Scientific) and reprobed with anti-lamin A/C antibody (1:5000 dilution). The proteins of interest were detected by a chemiluminescence substrate (SuperSignal West Femto; Thermo Scientific).
3
Evaluating Protein Expression in Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After three days of culture with or without TGF‐β1, cells were collected from six‐well plates of 5‐kPa, 30‐kPa PGS, and plastic by exposure to 5% trypsin/EDTA (Gibco) for 2–3 min at 37℃. After being washed with PBS (HyClone), cells were scraped and their total proteins were extracted in a Radio‐Immunoprecipitation Assay (RIPA) buffer (Fermentas; Thermo Fisher Scientific, Pittsburgh, PA, USA). Bicinchoninic acid (BCA) protein quantitative analysis kit (P0010S; Beyotime, Shanghai, China) was utilized to quantify the protein concentration in each sample. Briefly, the protein samples were loaded on pre‐cast 10% SDS‐PAGE (PG112; Epizyme, Shanghai, China) and transferred to polyvinyl difluoride (PVDF) membranes (Bio‐Rad). The membranes were incubated at 4℃ overnight with the primary antibodies, which are listed in Table 4 , and then incubated with HRP‐labeled secondary antibodies (Arigo; Hsinchu, Taiwan, China) for 1 h at room temperature. Then, the band images were developed with enhanced chemiluminescent (ECL) reagents (NCM Biotech; Suzhou, Jiangsu, China) and digitized on Image Quant LAS 4000 mini (GE Healthcare, Marburg, MA, USA). Image quantification was performed with Quantity One software (Bio‐Rad). GAPDH was used to serve as a loading control. All data were expressed as fold change in protein expression relative to the designated control group.
4
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Briefly, cells were collected and lysed with radioimmunoprecipitation assay buffer (RIPA) (50 mM Tris pH 7.4, 0.25% Na-deoxycholate, 1% NP-40, 150 mM NaCl and 1 mM EDTA) with proteinase and phosphatase inhibitors (Roche). Protein concentration was measured by Coomassie® Brilliant Blue (Bio-Rad). 20–30 μg total protein was separated by 8–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyl difluoride (PVDF) membranes (Bio-Rad) with a Trans-Blot® Turbo™ Transfer System. For membrane blotting, 5% non-fat dry milk in Tris-buffered saline with Tween 20 (TBST) was used. After incubation with primary antibodies in TBST (The details of the antibodies and the dilutions are listed in Additional file 1 : Table S2), the membranes were subsequently incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000 dilution, Bio-Rad). The signals were detected by the Clarity Western ECL substrate (Bio-Rad) and captured by ChemiDoc™ XRS+ Imaging Systems (Bio-Rad). Protein relative levels were quantified by analyzing the ratio of band intensity of target protein versus loading control using ImageJ.
5
Quantification of DRG Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were sacrificed by an overdose of chloral hydrate. Lumbar DRGs (L4-L6) from the ipsilateral side were quickly dissected out and frozen in liquid nitrogen. Each sample was lysed and centrifuged at 12,000 r/min for 30 min at 4℃ to collect the supernatant; 20 mg of protein was fractionated on 10% polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and then transferred to polyvinyl difluoride (PVDF) membranes (Bio-Rad, USA) for 2 h at 200 mA. The membranes were blocked in TBS (50 mM Tris-Base, 133 mM NaCl, pH = 7.4) containing a 5% dilution of non-fat milk powder for 2 h at room temperature (RT) and then incubated with primary antibody (anti-GRK6 at 1:500, Santa Cruz Biotechnology, USA; CXCR1 at 1:500, Santa Cruz Biotechnology, USA; anti-CXCR2 at 1:100, Boster, P.R. China; anti-CXCR4, Santa Cruz Biotechnology, USA; anti-GAPDH at 1:1000, Goodhere, China; anti-β-actin at 1:1000, Multi-Sciences, P.R. China) in TBS containing 1% milk at 4℃ overnight. After washing in TBST (0.5% Tween-20 in TBS), the PVDF membranes were incubated with HRP-conjugated secondary antibodies (1:4000, Multi Sciences Biotech Co., P.R. China) in TBS and 1% milk for 2 h at RT. Bands were visualized using ECL (Biological Industries, P.R. China) and exposed to Kodak X-ray films. Films were scanned and band intensities of target proteins were analyzed using Optic Quant software (ImageJ, NIH, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!