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Pvdf blot membrane

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The PVDF blot membrane is a widely used laboratory tool designed for protein transfer and detection in Western blotting applications. It is a thin, hydrophobic membrane made from polyvinylidene fluoride (PVDF) material. The PVDF blot membrane serves as a substrate for immobilizing proteins that have been separated by gel electrophoresis, allowing for further analysis and detection.

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Lab products found in correlation

Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions) Lipofectamine ltx reagent with plus reagent, by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Fluo 4am probe, by Thermo Fisher Scientific (1 mentions) Hek293t cell, by American Type Culture Collection (1 mentions) Qproteome ffpe tissue kit, by Qiagen (1 mentions) Anti gadph, by Cell Signaling Technology (1 mentions) Precision plus protein dual color standard, by Bio-Rad (1 mentions)

Close spelling variants of this product

PVDF (polyvinylidene difluoride) membranes for Western blots Trans-Blot Turbo Midi (PVDF) membranes Immu-Blot PVDF Membranes Immun-Blot PVDF Membranes for Protein Blotting Trans-blot PVDF membranes Transturbo blot PVDF membranes Immuno-Blot LF-PVDF membranes Trans-Blot Turbo mini 0.2 µm PVDF membranes Immun-Blot LF PVDF membrane Trans-Blot Turbo PVDF/Nitrocellulose membrane

4 protocols using pvdf blot membrane

1

Isolation and Purification of PMCA

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[γ-32P]ATP was obtained from Perkin-ElmerNEN Life Sciences (USA). HEK293T cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), Lipofectamine LTX Reagent with PLUS Reagent and cell culture supplements were purchased from Invitrogen-Thermo Fisher Scientific (Carlsbad, CA, USA). Antibiotic/antimycotic solution was obtained from Life Technologies Inc. (Carlsbad, CA, USA). Fluo-4-AM probe was obtained from Molecular Probes (Eugene, OR, USA). PVDF blot membrane was obtained from BioRad Laboratories (Reinach, Switzerland). Dimyristoyl phosphatidylcholine (DMPC) was obtained from Avanti Lipids (Alabaster, AL, USA). Thapsigargin (TG), (-)-epigallocatechin 3-gallate (EGCG), (-)-epicatechin, (+)-catechin, gallic acid, polyoxyethylene (10) lauryl ether (C12E10) and all the other chemicals used in this work were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human blood recently drawn at Fundación Fundosol (Buenos Aires, Argentina) was used for the isolation of PMCA. Donors provided informed consent for the donation of blood and for its subsequent legitimate use by the transfusion service.
Rinaldi D.E., Ontiveros M.Q., Saffioti N.A., Vigil M.A., Mangialavori I.C., Rossi R.C., Rossi J.P., Espelt M.V, & Ferreira-Gomes M.S. (2021). Epigallocatechin 3-gallate inhibits the plasma membrane Ca2+-ATPase: effects on calcium homeostasis. Heliyon, 7(2), e06337.
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2

Protein Extraction and Western Blot Analysis

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The total protein was extracted with Extraction Buffer EXB using Qproteome FFPE Tissue Kit following the instructions (Qiagen, San Diego, CA). Briefly, the excised tissues were incubated on ice for 5 min changing to 100°C for 20 min. The tissues were then incubated at 80°C for 2 h with agitation. After centrifugation, the supernatant containing the extracted proteins was transferred to a new Collection Tube and stored at −20°C. 50 μg of each extracted protein was used for PAGE. Proteins were fractionated by electrophoresis using 10% SDS-PAGE and electrophoretically transferred onto a PVDF blot membrane (Bio-Rad, Hercules, CA). Subsequent Western blot was performed as previously described [35 (link)].
Liu H., Gong M., French B.A., Liao G., Li J., Tillman B, & French S.W. (2015). Aberrant modulation of the BRCA1 and G1/S cell cycle pathways in alcoholic hepatitis patients with Mallory Denk Bodies revealed by RNA sequencing. Oncotarget, 6(40), 42491-42503.
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3

Protein Isolation from FFPE Tissue

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For protein isolation of FFPE tissue sections, paraffin was first removed from tissue samples as we previously described (Liu et al., 2014 (link)). Tissues samples were further pelleted and washed five times with RIPA buffer for 10 minutes at room temperature. The washed pellets were then suspended with reducing sample buffer quickly and denatured by heating at 100°C for 10 min. Then 50 µg of each cell lysate was used for PAGE. Proteins were fractionated by electrophoresis through 10% SDS-PAGE and electrophoretically transferred onto a PVDF blot membrane (Bio-Rad, Hercules, CA). Membranes were blocked with 5% BSA and TBST at room temperature for 2 h and washed three times with TBST. Anti-Ufm1 polyclonal antibodies were diluted according to the company’s recommendation in TBST and incubated with the membranes overnight at 4°C. After washes three times with TBST, the membranes were incubated with the corresponding secondary antibody. Proteins were visualized using the enhanced chemilumines-cence (ECL) detection system.
Liu H., Gong M., French B.A., Li J., Tillman B, & French S.W. (2014). Mallory-Denk Body (MDB) Formation Modulates Ufmylation Expression Epigenetically in Alcoholic Hepatitis (AH) and Non Alcoholic Steatohepatitis (NASH). Experimental and molecular pathology, 97(3), 477-483.
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4

Western Blot Analysis of PMCA Protein

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The collected cells were directly solubilized in sample buffer containing 150 mM Tris-HCl (pH 6.8), 4 % SDS, 5% DTT, 20% glycerol, urea (125 mg/ml) and bromophenol blue. The ladder was Precision Plus Protein™ Dual Color Standards (BioRad). Samples were separated using a 10% SDS gel (50 μg protein per lane), and proteins were transferred to a PVDF blot membrane (BioRad). After blocking, the blot was incubated with the anti-PMCA primary mouse monoclonal antibody 5F10 [41 (link)] (1:2000; Pierce) or anti-GADPH (1:1000, Cell signaling) overnight at 4 °C. Anti-mouse IgG, horseradish peroxidase-linked whole antibody (1:20000; Amersham, Buckinghamshire, UK) or anti-rabbit IgG, horseradish peroxidase-linked whole antibody (1:1000; Amersham, Buckinghamshire, UK) were used as the secondary antibody. Immunoreactive bands were visualized using the enhanced chemiluminescence method (Kalium Technologies, Buenos Aires, Argentina). Digital images were quantified using GelPro Analyzer version 4.0. Protein density was normalized to the GAPDH loading control.
Rinaldi D.E., Ontiveros M.Q., Saffioti N.A., Vigil M.A., Mangialavori I.C., Rossi R.C., Rossi J.P., Espelt M.V, & Ferreira-Gomes M.S. (2021). Epigallocatechin 3-gallate inhibits the plasma membrane Ca2+-ATPase: effects on calcium homeostasis. Heliyon, 7(2), e06337.
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