Following protein isolation and determination of concentration as described in the Western blot section, 20 µg of protein was transferred into each well to deposit on the nitrocellulose membrane (Sigma Aldrich, WHA10402506) via a Minifold I vacuum Dot-Blot system (Schleicher&Schuell, 10484138CP) [34 (link), 39 (link)], washed twice with 200 µL of PBS then dried for 60 min at 60 °C to adhere the proteins. The membranes were then cut and incubated in Ponceau S solution (Sigma, P7170) for protein visualization. The stain was removed and the membrane blocked for 1 h at room temperature with 5% milk in PBS-T. The membranes were incubated with antibodies against malondialdehyde (MDA)-positive proteins (Abcam, ab27642, 1:1000) as well as CD68 (Abcam, ab955, 1:1000), and interleukin 6 (Abcam, ab6672, 1:1000) overnight at 4 °C. The membranes were then treated in the same manner as those of Western blots.
Wha10402506
Manufactured by Cytiva
The WHA10402506 is a laboratory equipment product. It is designed for use in a laboratory setting. No further details about its core function or intended use can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
2 protocols using wha10402506
1
Dot-Blot Immunodetection of Oxidative Stress Markers
2
Dot-Blot Protein Quantification and Analysis
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One μl of plasma protein (approximately 30 μg of protein) was transferred into each well to deposit on the nitrocellulose membrane (Sigma Aldrich, WHA10402506) via a Minifold I vacuum Dot-Blot system (Schleicher&Schuell, 10484138CP) [14 (link),15 (link)], washed twice with 200 μl of PBS then dried for 60min at 60 °C to adhere the proteins. The membranes were then cut and incubated in Ponceau S solution (Sigma, P7170) for protein visualization and later normalization of the antibody staining. The stain was removed and the membrane blocked for 1 h at room temperature with 5% milk in PBS-T. The membranes were incubated with antibodies against caspase-3 (Cell Signaling, #9662S, 1:1000), thioredoxin interacting protein (TXNIP, Cell Signaling #14715, 1:1000), high-mobility-group-protein B1 (HMGB1, Cell Signaling #3935S, 1:1000), protease-activated receptor-1 (PAR1, BD Bioscience BD611522, 1:750), fibrinogen (Abnova #PAB5139 1:750) overnight at 4 °C. Positive bands were detected using ECL development (Thermo Fisher, 32 106, Millipore, ab5605, 1:750) and Chemilux Imager (CsX-1400 M, Intas). Densitometric quantification was performed using ImageJ software.
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