NHU cell cultures were lysed into electrophoresis sample buffer containing protease inhibitors and sonicated. Twenty micrograms of protein was resolved on 4–12% Bis-Tris NuPage™ polyacrylamide gels (Invitrogen) in 2-(N-morphilino) ethanesulfonic acid (MES) buffer and electro-transferred onto polyvinylfluoride membranes (Millipore). Membranes were blocked with Odyssey® blocking buffer (LI-COR Biotechnology OK Ltd., Cambridge, UK), incubated with primary antibodies for 16 h at 4 °C and bound antibody detected using Alexa Fluor® 680-conjugated anti-mouse secondary antibody (Invitrogen, Invitrogen supplied by Fisher Scientific UK Ltd., Loughborough, UK) or an IRDye 800-conjugated anti-rabbit secondary antibody (Tebu-Bio, Peterborough, UK). Antibody binding was visualized using an Odyssey® Sa Infrared Imaging System (LI-COR®). Protein quantification was performed using Odyssey® Image Studio™ software v5.0 (LI-COR®). Details of antibodies are given Table 2 and Table 3 .
Polyvinylfluoride membranes
Manufactured by Merck Group
Sourced in United States
Polyvinylfluoride membranes are a type of filtration membrane used in various laboratory applications. They are made from a synthetic polymer material and are designed to selectively filter and separate different substances based on their molecular size and properties. The core function of these membranes is to facilitate efficient filtration and separation processes in controlled laboratory environments.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey sa infrared imaging system, by LI COR (1 mentions)
Nupage bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Anti cytc, by Abcam (1 mentions)
Anti cleaved caspase 9, by Abcam (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti bax, by Proteintech (1 mentions)
Ec3 imaging system, by Analytik Jena (1 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Mouse anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Odyssey software, by LI COR (1 mentions)
Irdye goat anti mouse, by LI COR (1 mentions)
Irdye goat anti rabbit, by LI COR (1 mentions)
Irdye goat anti human, by LI COR (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
4 protocols using polyvinylfluoride membranes
1
Western Blot Analysis of Protein Expression
2
Protein Expression Analysis by Western Blot
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Cells were digested with pre-cooled RIPA lysate and protease inhibitors (Roche, Mannheim, BW, Germany) to extract total proteins and measure concentrations using the BCA method. SDS-PAGE was used to separate lanes containing 20 μg of proteins. Proteins were transferred to polyvinyl fluoride membranes (Millipore, Billerica, MA, USA) and then incubated in blocking buffer (5% skimmed milk) for 1 h. Membranes were then probed with anti-Bcl-2 (ProteinTech, 1:500), anti-Bax (ProteinTech, 1:500), anti-Cytc (Abcam, 1:500), anti-cleaved-caspase 9 (Abcam, 1:500) and anti-cleaved-caspase 3 at 4 °C overnight. The membranes were washed three times, each time for 10 minutes and then they were incubated in anti-rabbit IgG (Abcam, 1:5000) at room temperature for 45 minutes. The specific bands were revealed using the EC3 Imaging System (Analytik Jena US LLC, USA). Gel-Pro-Analyzer software was used to measure each band’s optical density.
3
Western Blot Analysis of CENPU and GAPDH
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MCF-7 cell lysates were prepared and electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel for separation of proteins, which were then electro-blotted onto polyvinyl fluoride membranes (Millipore). After being incubated in 5% non-fat dry milk [dissolved in Tris-buffered saline Tween (TBST)] for 1 h at ambient temperature, the membranes were probed with primary antibodies raised against our target proteins at 4°C overnight. After being rinsed with TBST for three times, the membranes were subjected to incubation in horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at ambient temperature. The protein bands were eventually developed by using the Enhanced Chemiluminescence Western Blotting Substrate kit (Pierce). The primary antibodies used in this study included rabbit anti-CENPU (diluted 500-fold; Abcam, ab117078) and mouse anti-GAPDH (diluted 2000-fold; Santa Cruz, sc-32233); the secondary antibodies used in this study included goat anti-rabbit IgG (diluted 2000-fold; Santa Cruz, sc-2004) and goat anti-mouse IgG (diluted 2000-fold; Santa Cruz, sc-2005).
4
Pv50 Protein Characterization by Western Blot
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The recombinant Pv50 and P. vivax schizont-stage parasite lysates were separated by 12% SDS-PAGE and then stained with Coomassie brilliant blue. For western blot analysis, recombinant proteins were electrophoretically transferred to polyvinylfluoride membranes (Millipore Corp., Billerica, MA, USA), and incubated with blocking buffer (5% non-fat milk in PBS containing 0.2% Tween 20, PBS/T) for 1 h at 37 °C. After blocking, penta anti-His antibody, mouse immune sera, rabbit immune sera, or mixed patient serum were diluted by 200-fold with PBS/T, and the specific quality of the His-tagged recombinant protein and immune serum was examined using the secondary antibody IRDye® goat anti-mouse (1:10,000 dilution), IRDye® goat anti-rabbit (1:20,000 dilution), or IRDye® goat anti-human (1:20,000) (LI-COR Biosciences, Lincoln, NE, USA). The fluorescence signals from the reaction were scanned on an Odyssey infrared imaging system (LI-COR Biosciences) and analyzed with Odyssey software (Li-Cor Biosciences).
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