JoVE Journal

Biology

This content is Open Access.

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

51.1K Views

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11:01 min

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November 20th, 2014

DOI :

10.3791/52099-v

November 20th, 2014

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Samantha L. Eaton¹, Maica Llavero Hurtado¹, Karla J. Oldknow², Laura C. Graham¹, Thomas W. Marchant¹, Thomas H. Gillingwater³^,⁴, Colin Farquharson², Thomas M. Wishart¹^,⁴

¹Division of Neurobiology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, ²Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, ³Centre for Integrative Physiology, University of Edinburgh, ⁴Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh

Summary

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

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Keywords Quantitative Fluorescent Western Blotting

Protein Detection

Fluorescent Labels

Comparative Expression Analysis

Protein Quantification

Troubleshooting Strategies

Chapters in this video

0:05

Title

1:32

Sample Preparation and Loading

3:43

iBlot2 Semi Dry Fast Protein Transfer

4:54

Post Transfer Membrane Handling

6:32

Visualization

7:51

Quantification of Scanned Proteins

8:50

Results: Representative Quantitative Fluorescent Western Blotting Experiments

10:31

Conclusion

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