Protein concentrations were determined by a Bradford protein assay. Sodium dodecyl sulfate polyacrylamidegel electrophoresis was performed to separate proteins from whole-cell and EV lysates, using M-PER mammalian protein extraction reagent (Thermo Fisher Scientific). After electrophoretic transfer to polyvinyl difluoridene membranes (Millipore), the membranes were incubated overnight at 4°C with polyclonal antibodies for tissue transglutaminase (tTG, Neomarkers, Fremont, CA, USA), and flotillin-2 (Cell Signaling Technology, Danvers, MA, USA), followed by horseradish peroxidase-linked secondary antirabbit (1:5000) or antimouse (1:10 000) secondary antibodies (Cell Signaling Technology). Antigen-antibody complexes were visualized by Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). For quantification of the data, films were scanned with a CanonScan 9950 F scanner (Ota, Tokyo, Japan) and images were analyzed using the public domain NIH ImageJ program (http://rsb.info.nih.gov/nih-image/ ).
Polyvinyldifluoridene membranes
Manufactured by Merck Group
Sourced in United States
Polyvinyldifluoridene (PVDF) membranes are a type of laboratory equipment used for filtration and separation purposes. These membranes are made from a thermoplastic polymer and are known for their chemical and thermal resistance, as well as their high mechanical strength. PVDF membranes can be used in a variety of applications, such as protein purification, cell culture, and sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Flotillin 2, by Cell Signaling Technology (4 mentions)
Horseradish peroxidase linked secondary anti rabbit or anti mouse secondary antibodies, by Cell Signaling Technology (3 mentions)
β actin, by Merck Group (2 mentions)
Horseradish peroxidase linked secondary antirabbit, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Thermo Fisher Scientific (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase linked anti rabbit or anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Zo 1 and zo 2, by Cell Signaling Technology (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
5 protocols using polyvinyldifluoridene membranes
1
Protein Expression Quantification for EVs
2
Western Blot Analysis of Protein Samples
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Protein concentrations were determined by Bradford protein assay (Thermo Fisher Scientific). Proteins in WCL and EVs lysates were separated through SDS PAGE and electrophoretically transferred to polyvinyldifluoridene membranes (Millipore, Billerica, MA and Bio-Rad, Hercules, CA). Membranes were incubated overnight at 4 °C with polyclonal antibodies for flotillin-2 (Cell Signaling Technology, Danvers, MA), Alix (Santa Cruz Biotechnology, Dallas, TX), AXL (Cell Signaling Technology), ZIKV envelop protein (GeneTex, Inc. Irvine, CA), as well as β-actin and GFAP (Sigma-Aldrich) followed by horseradish peroxidase-linked secondary anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology). Antigen-antibody complexes were visualized by Pierce ECL Western Blotting Substrate. For quantification of the data, films were scanned with a CanonScan 9950F scanner and images were analyzed using the public domain NIH image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/ ).
3
Protein Isolation and Western Blot Analysis
4
Western Blot Analysis of Protein Samples
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Protein concentrations were determined by Bradford protein assay. SDS PAGE separated proteins from whole cell and MVs lysates. After electrophoretically transferred to polyvinyldifluoridene membranes (Millipore, Billerica, MA and Bio-Rad, Hercules, CA). Membranes were incubated overnight at 4 °C with polyclonal antibodies for GAC (Dr. N. Curthoys, Colorado State University, Fort Collins, CO), Alix (Santa Cruz Biotechnology, CA) and flotillin-2 (Cell Signaling Technology, Danvers, MA), followed by horseradish peroxidase-linked secondary anti-rabbit or anti-mouse secondary antibodies (Cell signaling Technology). Antigen-antibody complexes were visualized by Pierce ECL Western Blotting Substrate. For quantification of the data, films were scanned with a CanonScan 9950 F scanner and images were analyzed using the public domain NIH image program (developed at the U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/ ).
5
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentrations were determined by Bradford protein assay. SDS PAGE separated proteins from the whole cell and EV lysates. Afterward, they were electrophoretically transferred to polyvinyldifluoridene membranes (Millipore, Billerica, MA and Bio-Rad, Hercules, CA). The membranes were incubated overnight at 4 °C with polyclonal antibodies for KGA and GAC (Dr. N. Curthoys, Colorado State University, Fort Collins, CO), tissue transglutaminase (tTG) (Lab Vision/Thermo, Fremont, CA), flotillin-2 (Cell Signaling Technology, Danvers, MA), and β-actin (Sigma), followed by horseradish peroxidase-linked secondary anti-rabbit or anti-mouse secondary antibodies (Cell Signaling Technology). Antigen-antibody complexes were visualized by Pierce ECL Western Blotting Substrate. For quantification of the data, films were scanned with a CanonScan 9950F scanner, and images were analyzed using the public domain NIH Image program (developed at the US National Institutes of Health).
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