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Immune blot pvdf membrane

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The Immune-Blot PVDF membrane is a polyvinylidene fluoride (PVDF) membrane designed for western blotting applications. It provides a stable and reliable platform for the detection and analysis of proteins.

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Lab products found in correlation

Pierce ecl western blotting substrate kit, by Thermo Fisher Scientific (3 mentions) Odyssey ir imaging system, by LI COR (3 mentions) Goat anti rabbit igg hrp, by Southern Biotech (3 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Novex, by Thermo Fisher Scientific (1 mentions) Enhanced chemi luminescence (ecl), by Merck Group (1 mentions) Dc protein assay kit, by Bio-Rad (1 mentions) Mini protein 3 cell, by Bio-Rad (1 mentions) Mini trans blot cell, by Bio-Rad (1 mentions) Ecl plus western blotting detection reagent, by GE Healthcare (1 mentions) Blocking solution, by Thermo Fisher Scientific (1 mentions) Anti mouse igg, by Merck Group (1 mentions) Mini protean tgx polyacrylamide gel, by Bio-Rad (1 mentions) Anti rabbit igg, by Merck Group (1 mentions) Ripa buffer, by Merck Group (1 mentions) Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions) Complete mini, by Roche (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Gradient polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)

27 protocols using immune blot pvdf membrane

1

Placental CD46 Expression Analysis

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Placental tissue lysates containing 20 μg protein were electrophoresed on 5–20% (w/v) polyacrylamide gradient gels (NOVEX, Life Technologies, USA) and transferred on to Immune-Blot PVDF membranes (Bio-Rad, USA). Expression levels of CD46 were probed using rabbit monoclonal antiCD46 antibody (ABCAM, USA). A secondary antibody goat anti-rabbit IgG-HRP (SouthernBiotech, USA) and pierce ECL Western blotting substrate kit (Thermo Fisher Scientific, USA) were used to develop the membranes. The Western bands were visualized using Odyssey IR imaging system (LI-COR Biosciences, USA).
Banadakoppa M., Balakrishnan M, & Yallampalli C. (2020). Common variants of fetal and maternal complement genes in preeclampsia: pregnancy specific complotype. Scientific Reports, 10, 4811.
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2

Robust Neuroscience Antibody Validation

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As detailed in the Results section, we took four independent steps to ensure quality of signal from our new neuroscience panel. One of these quality control steps involved western blots with conjugated antibodies. Only conjugated antibodies with Western blot banding pattern expected from the literature and unchanged from non-conjugated antibody were included. 34 of 36 antibodies passed this quality control (Table S1).
Appropriate ‘positive’ samples were homogenized in RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.2 by sonication (sonic dismembrator, Model F60, Supplier Fisher Scientific, 4+4 second at setting 15, with intermittent incubation on ice for 5 min). Protein lysates were separated in denaturing gels by electrophoresis transferred onto Immune-Blot PVDF membranes (Bio-Rad Cat#1620174) and blotted with metal-conjugated antibodies. Blots were developed by using ECL (Thermo Scientific, Cat # 1856135/6, 34094). We used P2 as the positive control for the CD11b signal because 5 micron filtration of P2 yielded synaptosomes without detectable microglia-derived particles.
Gajera C.R., Fernandez R., Postupna N., Montine K.S., Fox E.J., Tebaykin D., Angelo M., Bendall S.C., Keene C.D, & Montine T.J. (2018). Mass Synaptometry: High-Dimensional Multi Parametric Assay for Single Synapses. Journal of neuroscience methods, 312, 73-83.
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3

Western Blot Analysis of Iron Transporters

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Cells were immediately washed with ice-cold phosphate buffered saline (PBS) and lysed with lysis buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1 μg/mL aprotinin, 1% NP-40, 1% deoxycholic acid sodium salt and 100 μg/mL PMSF. Cell debris was removed by centrifugation (12,000 g at 4°C for 20 min). The protein concentration was determined using a DC Protein Assay Kit (Bio-Rad), and the protein samples were separated by SDS-PAGE, transferred to Immune-Blot PVDF membranes (Bio-Rad) and incubated at 4°C overnight with rabbit anti-DMT1 (divalent metal transporter 1); rabbit anti-TfR1 (transferrin receptor 1) (Santa Cruz). The membranes were then incubated at room temperature with rabbit anti-β-action antibodies (Santa Cruz) for 1 h and analyzed using the ECL (enhanced chemiluminescence) purchased from Sigma-Aldrich.
Shu T., Lv Z., Xie Y., Tang J, & Mao X. (2019). Hepcidin as a key iron regulator mediates glucotoxicity-induced pancreatic β-cell dysfunction. Endocrine Connections, 8(3), 150-161.
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4

CD133 Protein Expression in Huh7 Cells

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Western Blot was performed for CD133. Total protein of Huh7 was extracted by using cell lysis buffer. Twenty ug of total protein of Huh7 were size-separated, together with molecular weight standards (Fermentas) by (SDS–PAGE) on polyacrylamide gel, using a Mini Protein III Cell (Bio-Rad). After SDS–PAGE, proteins were electro-transferred with a semi-dry blotting system onto immune-blot PVDF membranes (Bio-Rad) using a Mini Trans-Blot Cell (Bio-Rad). Membrane was incubated overnight with anti-CD133 (clone W6B3C1) and with peroxidase-conjugated secondary antibody. Actin was used as a housekeeping protein. The peroxidase reaction was obtained by exposure of membrane in the ECL-Plus Western blot detection system solutions (ECL Plus Western blotting Detection Reagents, GE-Healthcare Bio-Sciences, Italia).
Sukowati C.H., Anfuso B., Fiore E., Ie S.I., Raseni A., Vascotto F., Avellini C., Mazzolini G, & Tiribelli C. (2019). Hyaluronic acid inhibition by 4-methylumbelliferone reduces the expression of cancer stem cells markers during hepatocarcinogenesis. Scientific Reports, 9, 4026.
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5

Western Blot Analysis of sFLT1 Protein

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Cell lysates containing 10μg protein were electrophoresed on 5–20% (w/v) polyacrylamide gradient gels (Novex, Life Technologies) and transferred on to Immune-Blot PVDF membranes (Bio-Rad). Expression of sFLT1 was probed with goat polyclonal human anti-FLT1 antibody (R&D system, USA). A secondary antibody rabbit anti-goat IgG-HRP (Southern Biotech, USA) and pierce ECL Western blotting substrate kit (Thermo Scientific, USA) were used to develop the membranes. The Western bands were visualized using Odyssey IR imaging system (LI-COR Biosciences, USA).
Banadakoppa M., Balakrishnan M, & Yallampalli C. (2018). Upregulation and release of soluble fms-like tyrosine kinase receptor 1 (sFLT1) mediated by complement activation in human syncytiotrophoblast cells. American journal of reproductive immunology (New York, N.Y. : 1989), 80(5), e13033.
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6

Western Blot Analysis of c-Kit Protein

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Total protein was extracted using RIPA buffer (Sigma, cat# R0278), containing a protease inhibitor cocktail (Complete Mini, Roche, cat# 04693124001). Equal amounts of proteins were denatured in loading buffer, separated on Mini-PROTEAN TGX polyacrylamide gels (Bio-Rad, cat# 456–9034) and electro transferred into Immune-Blot PVDF membranes (Bio-Rad, cat# 162–0177). The membranes were blocked with blocking solution (Invitrogen, cat# 000105) and incubated with primary antibodies against c-Kit (rabbit monoclonal; Cell Signaling: 1:1000, cat# 3074S), followed by peroxidase-conjugated secondary antibodies (anti-rabbit IgG; Sigma: 1:10000, cat# A6667, or anti-mouse IgG; Sigma: 1:1000, cat# A4416, respectively). Amersham ECL prime western blotting detection reagent (GE Healthcare, cat# RPN2236) was used and the chemi-luminescent signal was detected using a Bio-rad ChemiDoc XRS+ (Bio-rad). Data are representative of three independent experiments.
Bulatovic I., Ibarra C., Österholm C., Wang H., Beltrán-Rodríguez A., Varas-Godoy M., Månsson-Broberg A., Uhlén P., Simon A, & Grinnemo K.H. (2015). Sublethal Caspase Activation Promotes Generation of Cardiomyocytes from Embryonic Stem Cells. PLoS ONE, 10(3), e0120176.
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7

Quantifying Crry and C3 Expression

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Tissue lysates containing 20μg protein were electrophoresed on 5–20% (w/v) polyacrylamide gradient gels (Thermo Fisher, Grand Island, NY, USA) and transferred on to Immune-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). Expression levels of Crry and C3 were probed using rabbit monoclonal antiCD46 antibody (Abcam, Cambridge MA, USA, cat# 108307). A secondary antibody goat anti-rabbit IgG-HRP (Southern Biotech, USA) and pierce ECL Western blotting substrate kit (Thermo Fisher) were used to develop the membranes. The Western bands were visualized using Odyssey IR imaging system (LI-COR Biosciences, Lincoln, NE, USA).
Banadakoppa M., Pennington K., Balakrishnan M, & Yallampalli C. (2020). Complement inhibitor Crry expression in mouse placenta is essential for maintaining normal blood pressure and fetal growth. PLoS ONE, 15(8), e0236968.
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8

Bladder Protein Expression Analysis

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Bladder specimens were homogenized in protein extraction solution (T-PER; Thermo Scientific, USA) with proteinase and phosphatase inhibitor cocktail (Roche Applied Science, Penzberg, Germany). The standard techniques for immunoblotting were followed. Briefly, 10 μg of bladder protein extract were electrophoresed at a constant voltage of 150 V for 2 h and were then transferred to Immune-Blot PVDF Membranes (Bio-Rad, Hercules, CA, USA). The membranes were immunoblotted overnight at 4°C with the primary antibody. The antibodies used were as follows: rabbit anti-human SV2A antibody (Abcam, Cambridge, UK), goat anti-human SNAP-25 antibody (Abcam, Cambridge, UK), and rabbit anti-human P2X3 antibody (Santa Cruz Biotechnology, USA). After washing the membranes, they were incubated with a secondary antibody for 2 h at room temperature. The amount of glyceraldehyde phosphate dehydrogenase was calculated as the internal control. Quantitative analysis was performed using TotalLab Quant software V13.2 (TotalLab Ltd. UK).
Liu H.T., Chen S.H., Chancellor M.B, & Kuo H.C. (2015). Presence of Cleaved Synaptosomal-Associated Protein-25 and Decrease of Purinergic Receptors P2X3 in the Bladder Urothelium Influence Efficacy of Botulinum Toxin Treatment for Overactive Bladder Syndrome. PLoS ONE, 10(8), e0134803.
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9

Western Blot Analysis of Dental Stem Cells

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Proteins were collected from DPSCs using Pierce RIPA buffer (Invitrogen). The proteins were subjected to 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and subsequently transferred onto Immune-Blot PVDF membranes (Bio‐Rad Laboratories). After blocking with TBST containing 2% BSA or 5% dry skim milk (Yukijirushi, Tokyo, Japan) for 1 h at room temperature, the membranes were reacted with the anti-TH antibody (1:1000) or anti-β-actin antibody (1:1000) overnight at 4 °C. Then, the membranes were stained with biotinylated anti‐rabbit IgG (Nichirei Biosciences) or anti-mouse IgG (Nichirei Biosciences) and reacted with an avidin-peroxidase conjugate (1:2000, Sigma-Aldrich). Reactive bands were detected using the ECL select Western blotting detection system (GE Healthcare, Buckinghamshire, UK). Images were analyzed by Image Quant LAS 4000 (GE Healthcare) and ImageJ 1.53e (Java 1.8.0_172, http://imagej.nih.gov/ij).
Fujino S., Hamano S., Tomokiyo A., Sugiura R., Yamashita D., Hasegawa D., Sugii H., Fujii S., Itoyama T., Miyaji H, & Maeda H. (2023). Dopamine is involved in reparative dentin formation through odontoblastic differentiation of dental pulp stem cells. Scientific Reports, 13, 5668.
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10

Gata5 Protein Expression Analysis

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After transfection of wildtype and mutant Gata5 tagged expression constructs into HeLa cells, 20 micrograms of cell lysate was loaded per lane and separated using 10% SDS-acrylamide gels, and transferred to immune-blot PVDF membranes (Bio-rad, Hercules, CA). After blocking with 5% non-fat milk in PBST, the membrane was probed with primary monoclonal mouse anti-Xpress (1:1000, R910-25, Life Technologies, Grand Island, NY) and mouse anti-GAPDH antibody (1:5000, NB300-221, Novus Biologicals, Littleton, CO). The membrane was further probed with horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG (PI-2000).
Bonachea E.M., Chang S.W., Zender G., LaHaye S., Fitzgerald-Butt S., McBride K.L, & Garg V. (2014). GATA5 Sequence Variants Identified in Individuals with Bicuspid Aortic Valve. Pediatric research, 76(2), 211-216.
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