First, radioimmunoprecipitation assay lysis buffer was utilized for cell lysis, and after 15 min of centrifugation at 12,000 revolutions/min, the total protein concentration was assessed with bicinchoninic acid/protein detection kit (Thermo Scientific, New York, United States); 25 μg proteins were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Afterward, proteins were transferred to polyvinylidene fluoride (Bio-Rad Laboratories, Inc., United States) membrane, which was sealed with 5% skimmed milk for 1 h at room temperature and cultured overnight with primary antibodies at 4°C. Antibodies were all bought from Abcam. Primary antibodies were all rabbit antibodies: anti-CREB1 antibody, anti-PSEN1 antibody, anti–β-actin antibody, anti–E-cadherin antibody, anti–N-cadherin antibody, and antivimentin antibody. Secondary antibody was goat anti-rabbit immunoglobulin G (IgG) H&L (HRP).
Polyvinylidene fluoride (pvdf)
Manufactured by Bio-Rad
Sourced in United States, China, Germany
PVDF (Polyvinylidene Fluoride) is a type of membrane material used in various laboratory applications. It is a durable, chemically resistant, and hydrophobic polymer that is commonly used in Western blotting techniques for protein detection and analysis. The primary function of PVDF membranes is to serve as a substrate for the immobilization of proteins, allowing for their separation, transfer, and subsequent detection.
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Lab products found in correlation
Bca protein assay kit, by Beyotime (14 mentions)
Ripa buffer, by Merck Group (12 mentions)
Image lab software, by Bio-Rad (10 mentions)
Odyssey imaging system, by LI COR (10 mentions)
Anti β actin, by Abcam (9 mentions)
Odyssey v2, by LI COR (9 mentions)
β actin, by Santa Cruz Biotechnology (9 mentions)
Sds page, by Bio-Rad (9 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (8 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Chemidoc xrs system, by Bio-Rad (7 mentions)
Chemidoc xrs imaging system, by Bio-Rad (7 mentions)
Bradford assay, by Bio-Rad (7 mentions)
Ripa buffer, by Thermo Fisher Scientific (7 mentions)
β actin, by Merck Group (6 mentions)
Chemidoc, by Bio-Rad (6 mentions)
Protease inhibitor cocktail, by Merck Group (6 mentions)
β actin, by Cell Signaling Technology (6 mentions)
338 protocols using polyvinylidene fluoride (pvdf)
1
Protein Expression Analysis by Western Blot
2
Western Blot Protein Analysis
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Proteins were extracted by homogenizing in RIPA buffer (Sigma-Aldrich, USA) with phenylmethanesulfonyl fluoride and phosphatase inhibitor (Bimake, USA). Proteins were separated by SDS-PAGE gel (Bio-Rad, USA) and then transferred to polyvinylidene fluoride (Bio-Rad) membranes. Antibodies are listed in Supplementary Table 1 (available online). After incubating the antibody, the membrane was washed, and the band was observed by the ECL reaction (Bio-Rad) for appropriate exposure. Quantify with ImageJ software (version 1.41)
3
Quantification of Aβ Levels in C. elegans
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Rabbit polyclonal Aβ1-42 primary antibodies were from Abcam (ab39377). For Western blot analysis, CL2006 worms were synchronized by allowing 10-15 hermaphrodites to lay eggs overnight on OP50 seeded NGM plates at 16°C. The parents were removed, and eggs were allowed to hatch and develop to the L4 stage. Subsequently, worms were transferred to RJ (2mg/mL) / eRJ (1mg/mL) containing NGM plates and continued to grow at 20°C. CL2006 worms on regular NGM plates without RJ/eRJ served as control. After 10 days of growth, worms were transferred to micro-centrifuge tubes and washed with S-basal followed by protein immobilization on polyvinylidenefluoride (Bio-Rad) membrane. polyvinylidenefluoride membrane was incubated with primary antibodies (1:1,000) diluted in 5% nonfat dry milk and then with secondary, HRP-conjugated goat anti-rabbit antibodies (Genscript, A00098; diluted 1:10,000). ACTIN was used as loading control, and the anti-ACTIN antibodies (MAB1501) were from EMD Millipore. Detection was undertaken with standard ECL protocol. Mean intensity of Aβ signals was analyzed using ImageJ software (National Institute of Health).
4
Western Blot Analysis of ANO1 and ANO2
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The thalamus and kidney from BALB/c mice, as well as ANO2-EGFP-transfected and untransfected NIH/3T3 cells, were lysed in RIPA buffer (Tris-base, 6.5 mM; NaCl, 15 mM; EDTA, 1 mM; NP-40, 1%; and Na-deoxycholate, 0.25%) with 1 mM phenylmethyl sulphonyl fluoride, Na3VO4, NaF and protease cocktail. Lysates were centrifuged at 13,000 r.p.m. for 10 min at 4 °C, and the supernatants were isolated for SDS–PAGE. From each sample, 50 μg of protein was resolved on 8% polyacrylamide gels and subsequently electroblotted to polyvinylidene fluoride (Bio-Rad, USA) membranes. Blots were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibodies. Signals were visualized using ECL Plus reagent (Thermo Scientific, USA). The following antibodies were used: rabbit anti-ANO1 (ab53212, 1:500, Abcam, UK), rabbit anti-ANO2 (1:500 (ref. 35 (link))), mouse anti-β-actin (sc-47778, 1:2,000, Santa Cruz, USA), goat anti-rabbit-HRP-conjugated (31460, 1:5,000, Pierce, USA) and goat anti-mouse-HRP-conjugated (31430, 1:5,000, Pierce).
5
Comprehensive Analytical Techniques for Protein Expression
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TRIzol reagent, DNaseI enzyme, Moloney Murine Leukemia Virus reverse transcriptase, and SYBR Green positron emission tomography Master Mix were purchased from Invitrogen (Carlsbad, CA, USA). The BCA protein analysis reagent kit was provided by Pierce (Rockford, USA). Polyvinylidene fluoride was provided by Bio-Rad Laboratories (Hercules, USA). Rabbit anti-human osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were purchased from Novus Biologicals (Littleton,USA), rabbit anti-human receptor activator of nuclear factor-κB (RANK), β-Actin as well as goat anti-rabbit IgG horseradish peroxidase were provided by Cell Signaling Technology (Boston, USA). The ECL reagent kit was purchased from Amersham Pharmacia Biotech (Piscataway, USA), and all other reagents were of analytical grade.
6
TK Extract Modulates MAPK Signaling
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HaCaT cells (3×105 cells per well) were seeded into 6-well plates and incubated for 24 h. The medium was replaced with a serum-free medium containing indicated concentrations of TK extract (0, 1, 10, and 100 μg/mL), and the cells were incubated for an additional 24 h. A total of 20 μg of whole-cell lysate proteins were loaded in each lane of 6–10% of acrylamide SDS-PAGE gel. Antibodies for p38 (sc-535), JNK1/2 (sc-7345), p-JNK1/2 (sc-6254), ERK5 (sc-398015), p-ERK5 (sc-135760), GAPDH (sc-25778), and goat anti-rabbit IgG-HRP secondary antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for p-p38 (#9211), ERK1/2 (#9102), p-ERK1/2 (#9101), AKT (#9272), and p-AKT (9271) were purchased form Cell Signaling Technology (Danvers, MA, USA), and goat anti-mouse IgG-HRP secondary antibody was purchased from Bio-Rad (Hercules, CA, USA). Polyvinylidene fluoride (Bio-Rad, Hercules, CA, USA) membrane and bovine serum albumin (Roche, Basel, Switzerland) were used. The membranes were developed using enhanced ECL (Bio-Rad, Hercules, CA, USA) on a UVITEC imaging system (UVITEC, Cambridge, UK) [22 (link)].
7
Western Blot Analysis of Striatal Proteins
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The striatum of mice was lysed in CelLytic™ M cell lysis reagent (Sigma-Aldrich, C3228, St Louis, MO, U.S.A) containing protease inhibitors (Sigma-Aldrich, P8340, St Louis, MO, U.S.A) and phosphatase inhibitors (Sigma-Aldrich, P0044, St Louis, MO, U.S.A). The protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed after loading equal amounts of protein into each lane. The proteins were transferred to polyvinylidene fluoride (Bio-Rad) membranes for immunoblotting. After the membranes were blocked with bovine serum albumin (BSA) (Sigma-Aldrich, A9647, St Louis, MO, U.S.A), they were blotted by adding primary antibodies against DAT (Merck, AB1591P, Darmstadt, Germany) or GAPDH (Genetex, GTX100118, Hsinchu, Taiwan) followed by the appropriate secondary antibodies. The protein bands were detected using an enhanced chemiluminescence kit (PerkinElmer, Waltham, MA) and quantified using 1Dscan Ex gel analysis software (Scanalytics).
8
Exosome Protein Profiling by Western Blot
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Exosomes and cells were lysed using radioimmunoprecipitation assay lysis buffer containing protein inhibitors. An equivalent quantity of proteins (15 μg) was electrophorized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and then transferred to a polyvinylidene fluoride (Bio-Rad Laboratories, Inc) membrane. After being blocked in 5% (w/v) skimmed milk, the membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies were: FRMD3 (ab166071, 1:1000), CD-9 (ab92726, 1:2000), CD-63 (ab134045, 1:10000), TSG101 (ab125011, 1:1000), E-cadherin (ab40772, 1:10000), N-cadherin (ab76011, 1:5000), Vimentin (ab92547, 1:1000) and GAPDH (ab9485, 1:2500). Subsequently, the membrane was reacted with horseradish peroxidase-labeled goat anti-rabbit secondary antibody IgG H&L (ab6721, 1:2000) for 1h. All antibodies were bought from Abcam Company (UK). All bands were measured by electrochemiluminescence (ECL) western blot kit (Amersham Biosciences, Little Chalfont, UK) and processed by Image Lab 6.0.1 Software (Bio-Rad Laboratories, Inc.).
9
Protein Extraction and Western Blot
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Total protein from organoid cultures or tissues was isolated using RIPA buffer (Sigma, USA). Lysates were centrifuged and separated on a gradient gel (8–12%) via SDS–polyacrylamide gel electrophoresis, blotted onto a membrane (polyvinylidene fluoride; Bio Rad, USA), and analysed using specific antibodies (Supplementary Table 3 ). Bands were visualized using enhanced chemiluminescence (ECL Kit, Amersham Biosciences, USA). Images of all uncropped western blots can be found in Supplementary Fig. 11 .
10
Western Blot Analysis of Cell Lysates
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Cell lysates were washed with PBS and lysed in cold RIPA supplemented with a cocktail of protease and phosphatase inhibitor (Roche) on ice. Protein concentrations were determined by a bicinchoninic acid (BCA) method using Pierce BCA protein Assay (Thermo Fisher Scientific). An equal quantity of samples mixed with sodium dodecyl sulfate (SDS)-containing sample buffer were boiled at 95 °C for 5 min and separated by SDS-poly acrylamide gel electrophoresis. Proteins were transferred to polyvinylidene fluoride for immunoblotting (Bio-rad, Hercules, CA, USA). The membrane was blocked with 5% skim milk in TBS. Primary antibodies diluted in tris-buffered saline and Tween 20 (TBS-T) were incubated overnight at 4 °C. The following antibodies were used: anti-ZO1 (Thermo Fisher Scientific), anti-DSG2 (Abcam), and anti-β-Actin (Santa Cruz, CA, USA). Following overnight incubation, the membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h (Santa Cruz) diluted in TBS-T. The membrane was washed, and proteins were detected using an enhanced chemiluminescence reagent (Pierce, Thermo Fisher Scientific).
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