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Western Blotting Protocol
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In this video we take you through all the steps in our Western Blot (WB) Protocol so you can replicate our procedure and get reproducible and reliable results.
Contents:
0:00 Introduction
1:06 Solutions and Reagents
2:11 Sample Preparation and Protein Blotting
4:33 Membrane Blocking and Antibody Incubations
6:07 Protein Detection
6:45 Importance of Following the Recommended Protocol
8:37 Conclusion
Western blotting is a widely used immunoassay used by research scientists to monitor expression of a specific protein in a complex cell or tissue extract. It utilizes antibodies that recognize a specific protein of interest or a post-translational modification like phosphorylation, acetylation, methylation, and ubiquitination. At Cell Signaling Technology (CST), we perform thousands of Western blots daily using a protocol that has been optimized for over a decade to develop and validate antibodies with exceptional specificity, sensitivity, and reproducibility.
We first list all the the solutions and reagents you’ll need. Then, we give detailed descriptions on how to prepare the sample, perform the protein blot and immunoassay, and detect proteins chemiluminescently. Finally, we describe critical experimental steps in Western blotting and explain how small changes to the protocol, like changing incubation times or dilution buffers, can affect the final outcome of your blot.
Contents:
0:00 Introduction
1:06 Solutions and Reagents
2:11 Sample Preparation and Protein Blotting
4:33 Membrane Blocking and Antibody Incubations
6:07 Protein Detection
6:45 Importance of Following the Recommended Protocol
8:37 Conclusion
Western blotting is a widely used immunoassay used by research scientists to monitor expression of a specific protein in a complex cell or tissue extract. It utilizes antibodies that recognize a specific protein of interest or a post-translational modification like phosphorylation, acetylation, methylation, and ubiquitination. At Cell Signaling Technology (CST), we perform thousands of Western blots daily using a protocol that has been optimized for over a decade to develop and validate antibodies with exceptional specificity, sensitivity, and reproducibility.
We first list all the the solutions and reagents you’ll need. Then, we give detailed descriptions on how to prepare the sample, perform the protein blot and immunoassay, and detect proteins chemiluminescently. Finally, we describe critical experimental steps in Western blotting and explain how small changes to the protocol, like changing incubation times or dilution buffers, can affect the final outcome of your blot.
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