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:
0:27 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
14:00 StarBright fluorescent secondary antibodies
16:00 Current practice demands multiplexing
16:32 Why fluorescence
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
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