Western blot analysis was performed using cell lysates prepared in T-PER tissue protein extraction reagent (Thermo) with protease and phosphatase inhibitor cocktail (Roch). After resolved by SDS-PAGE (4–20% TGX gradient gel, BioRad), proteins were transferred to PDMF membrane (Bio-Rad), rinsed in PBST, blocked in 5% dry milk, and incubated with a rabbit monoclonal antibody against mouse β3-Integrin (at 1:1000 dilution, #13166; Cell Signaling Technology) and a mouse monoclonal antibody for GAPDH (at 1:1000 dilution, #RDI-TRK5G4-6C5; Research Diagnostics Inc). HRP-conjugated goat anti-rabbit secondary antibody (1:3000, Bio-Rad), and goat anti-mouse secondary antibody (1:3000, Invitrogen) were used for detection by chemiluminescence (SuperSignal WestPico PLUS; Thermo). Signals were obtained by a LICOR-Odyssey scanner.
Pdmf membrane
Manufactured by Bio-Rad
The PDMF membrane is a porous polymer membrane designed for use in laboratory applications. It is made of polydimethylsiloxane (PDMS) and serves as a filtration medium. The membrane has a defined pore size that allows for the separation and isolation of specific components from liquid samples.
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Lab products found in correlation
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Integrin β3, by Cell Signaling Technology (1 mentions)
Tgx gradient gel, by Bio-Rad (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Bio-Rad (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Laemmli sds sample buffer, by Thermo Fisher Scientific (1 mentions)
Rabbit anti v5, by Cell Signaling Technology (1 mentions)
2 protocols using pdmf membrane
1
Quantifying Integrin Expression in Cells
2
Immunoprecipitation of Epitope-Tagged Proteins
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Cell lysates from transfected 293T were incubated with anti-FLAG M2 magnetic beads (Millipore Sigma) or anti-V5 agarose beads (Millipore Sigma) at 4°C for 4 h or overnight. Beads were then washed 3 times with NP-40 cell lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40 supplemented with 0.5 mM dithiothreitol) and 1 time with PBS. 5% input and immunoprecipitated fractions were boiled in Laemmli SDS sample buffer (Thermo). Protein samples were processed for SDS-PAGE (4–15% gel; Bio-Rad), transferred to PDMF membrane (Bio-Rad) for detection using rabbit anti-HA, rabbit anti-V5, or rabbit anti-FLAG antibodies (Cell Signaling), followed by HRP-conjugated goat anti-rabbit antibodies (Bio-Rad). ECL substrate (Thermo Fisher Scientific) was used for detection. Exposure and images were performed using LI-COR Fc imager (LI-COR biosciences).
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