Cells and brain tissue were dissolved with sodium dodecyl sulfate (SDS) (Amresco, Solon, OH, USA) buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, and 50 mM dithiothreitol). The proteins were then transferred to polyvinylidene difluoride (PVDF) (Amresco, Solon, OH, USA) membranes. The blots were blocked in 4% bovine serum albumin (BSA) (Amresco, Solon, OH, USA) in Tris-buffered saline tween-20 (TBST) (Amresco, Solon, OH, USA) solution for 30 min at room temperature and then incubated at 4° C overnight with primary antibodies. After incubation with secondary antibodies at room temperature for 1 h, the blot was visualized using the Chemi Doc XRS imaging system (Bio-Rad, Hercules, CA, USA).
Polyvinylidene fluoride (pvdf)
Manufactured by Avantor
Sourced in United States
PVDF (Polyvinylidene Fluoride) is a type of fluoropolymer material used in various laboratory equipment and applications. It is known for its chemical resistance, thermal stability, and mechanical strength. PVDF is commonly used in the construction of components such as tubing, valves, and other laboratory hardware that require these properties.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Avantor (2 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Bca detecting kit, by Keygen Biotech (1 mentions)
Fibronectin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Cell Signaling Technology (1 mentions)
E cadherin, by Abcam (1 mentions)
Ecl western blotting substrate kit, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti rabbit igg antibody, by Merck Group (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Ecl prime detection system, by Thermo Fisher Scientific (1 mentions)
6 protocols using polyvinylidene fluoride (pvdf)
1
Western Blot Analysis of Brain Proteins
2
Western Blot Analysis of HDAC4
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Total proteins were harvested using radio immunoprecipitation assay (RIPA) lysis buffer (Sigma) and qualified by a BCA detecting kit (Keygen, Nanjing, Jiangsu, China) according to the manufacturer's protocol. Protein samples were subjected to 10% SDS‐PAGE and transferred onto a PVDF (Amresco, Washington, DC, USA) membrane and then incubated with anti‐HDAC4 antibody (Abcam, Cambridge, MA, USA; dilution rates of 1:500) at 4°C overnight. The membranes were incubated with secondary antibodies (Abcam, dilution rates of 1:2000) at 25°C for 1 hour on the following day. Protein bands were detected on X‐ray film using an enhanced chemiluminescence detection system.
3
Western Blot Analysis of Neuronal and Glial Markers
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Cells were dissolved with sodium dodecyl sulfate (SDS; Amresco, Solon, OH, USA) buffer (62.5 mM Tris–HCl, 10% glycerol, 2% SDS, and 50 mM dithiothreitol). The proteins were then transferred to polyvinylidene difluoride (PVDF) (Amresco) membranes. The blots were blocked in 4% bovine serum albumin (Amresco) in Tris-buffered saline/Tween-20 (Amresco) solution for 30 min at room temperature and then incubated at 4 °C overnight with the following primary antibodies: mouse monoclonal anti-P75 (Sigma-Aldrich), mouse monoclonal anti–glial fibrillary acidic protein (GFAP; Sigma-Aldrich), human monoclonal anti-HIF-1α (Sigma-Aldrich), human monoclonal anti-III beta-tubulin (Sigma-Aldrich), human monoclonal anti-TH (Sigma-Aldrich), human monoclonal anti-GFAP (Sigma-Aldrich), human monoclonal anti-nuclear receptor related 1 protein (Nurr1; Sigma-Aldrich), human monoclonal anti-pituitary homeobox 3 (Pitx3; Sigma-Aldrich), human monoclonal anti-Lmx1b (Sigma-Aldrich), and human monoclonal anti-actin (Sigma-Aldrich). After incubation with secondary antibodies at room temperature for 1 h, the blot was visualized using ChemiDoc XRS imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
4
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Total protein of tissue specimens and cells were lysed by ice-cold radio immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, Merck KGaA). Following protein concentration quantification with a Bicinchoninic Acid protein assay kit (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), 40 µg protein samples were subjected to 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (PVDF; Amresco, LLC, Solon, OH, USA) and then blocked by 5% BSA (Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. Subsequently, membranes were incubated with antibodies against SMAD4 (1:5,000; cat no. ab40759; Abcam, Cambridge, MA, UK), E-cadherin (1:100; cat. no. ab76055; Abcam), fibronectin (1 µg/ml; cat. no: ab23750; Abcam) and GAPDH (1:10,000; cat. no. ab128915; Abcam) at 4°C overnight. The following day, the membranes were incubated with secondary antibodies [goat anti-rabbit IgG horseradish peroxidase (HRP), 1:2,000; cat. no. ab205718, Abcam] at room temperature for 1 h. Following washing with TBST 3 times, an ECL Western Blotting Substrate kit (cat. no. ab65623; Abcam) was used for chemiluminescence imaging with Image J software version 2X (National Institutes of Health, Bethesda, MD, USA).
5
Examination of Citrullinated Proteins
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Our group has developed a method for the examination of citrullination26 (link), which can be used for the identification of citrullinated proteins in biological samples. Saliva samples corresponding to 20 µg total salivary protein were used for dot-blot analysis. The PVDF (VWR International Ltd., Radnor, Pennsylvania, USA) membrane was activated by methanol and the samples were dropped onto the membrane in duplicates. Blocking was carried out using 1% polyvinylpirrolidone PVP-40 in TTBS buffer (10 mM Tris, 150 mM NaCl, pH 8.0) for 60 min at room temperature27 (link). The membrane was incubated with 1:15,000 anti citrulline mouse polyclonal antibody (PAB0672, Covalab UK Ltd. Cambridge, UK) at 4 °C overnight, followed by incubation with 1:15,000 horse radish peroxidase (HRP)—conjugated anti-rabbit IgG antibody (Sigma-Aldrich, St. Louis, USA) for 1 h at room temperature. The membrane was visualized using ECL substrate detection kit (Thermo FisherScientific Inc, Waltham, MA, USA) and developed on autoradiographic film (Agfa, Motsel, Belgium). Densitometry was carried out by using QuantityOne 4.6.7 software (BioRad Ltd., Hercules, CA, USA) and individual background substraction was applied. Density values are presented relative to the average density of 5 parallel ferryl (IV)-hemoglobin reference spots.
6
Immunoblotting Analysis of Eaat2a and Gapdh
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Total protein collection and immunoblotting techniques were performed as previously described [32 (link)], with minor modifications. Five cerebellums were pooled for each experimental replicate. Both chicken anti-Eaat2a (1:1000, gift of Neuhauss lab) [33 (link)] and rabbit anti-Gapdh (1:2500; cat #: ab210113, Abcam, Cambridge, UK) polyclonal anti-sera were incubated on membranes (0.45 µm PVDF, VWR, Radnor, PA, USA) that were blocked overnight at 4 °C in 1 × TBS/5% nonfat dry milk/0.1% Tween 20. Membranes were washed 4 × 15 min in 1 × TBS/0.1% Tween 20. The membranes were incubated with anti-chicken or anti-rabbit peroxidase secondary antibody (1:10,000, chicken, cat #: A9046, Sigma-Aldrich, St. Louis, MO, USA; rabbit, NA934, VWR International, Radnor, PA, USA), washed, and detected as described previously [32 (link)]. The blot was incubated with the ECL-Prime detection system (Fischer Scientific, Waltham, MA, USA) and exposed to X-ray film.
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