Immunoblotting (Western) Protocol
Scott Vermilyea
Published: 2024-03-21 DOI: 10.17504/protocols.io.5qpvo3e3bv4o/v1
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Abstract
This protocol details the western blot technique.
Attachments
Western Blot and Lysate Prep Protocol.docx
Steps
Immunoblotting (Western)
1.
Run protein lysates using desired Criterion™ TGX™ gels.
2.
Transfer onto nitrocellulose membranes.
3.
Detect protein of interest using appropriate primary antibodies at 4°C 1h 0m 0s, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1h 0m 0s at Room temperature.
4.
To visualize, use chemiluminescent substrates and image using ImageQuant LAS 4010 detection system.
5.
Perform densitometry on Western blot images using ImageQuant TL 8.1 software.
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