Quantitative Western Blotting: How to improve your data quality and reproducibility

Learn how to improve your western blotting results and generate publishable data. Poulomi Acharya, PhD walks you through techniques to improve the detection and quantification of proteins using western blotting.

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Quick links to Chapters:
0:01 Introduction
0:27 Western blotting is a widely used, powerful technique
1:20 The first western blot
2:00 Steps of western blotting
2:18 Sample preparation (cell/tissue lysate)
2:52 Sample preparation (lysis technique)
4:15 Choosing the right gel/buffer chemistry
5:53 Choosing the right gel percentage
6:13 Choosing the right protein standards
6:44 Protein loading best practices
8:04 Running the gel
8:24 Protein Transfer - Selecting the membrane
9:38 Best practices for transfer
10:35 Wet vs semi-dry transfer
11:35 Blocking:blocking agent, buffer, detergent
12:12 Antibodies
13:15 Immunodetection
14:00 StarBright fluorescent secondary antibodies
16:00 Current practice demands multiplexing
16:32 Why fluorescence
18:00 Stripping/Re-probing
19:02 Data analysis
19:47 Who is a better high-jumper?
20:22 Challenge: Linear range and saturation
20:51 Journals recommend total protein normalization
22:30 Linear Quantitation
23:40 Conclusion

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