Western Blot: How to do a Wet Transfer
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2 سال پیش
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The western blot is a
The western blot is a widely used research technique that detects proteins using antibodies. To conduct a western blot, the proteins need to be transferred onto a membrane. There are different ways to transfer proteins onto the membrane. This video will be focusing on the wet transfer method.
The video lists the materials and reagents needed for a wet transfer and how to conduct it. Note that there are several different kinds of transfer buffers used. One of the most common is the Towbin transfer buffer, named after one of the scientists who helped develop the western blot. It contains 25mM of Tris, 19.2 mM of glycine and 20% methanol.
This video was created by Kieran Anderson, Kevin Cao, Aniqa Chaudhry, Brooke D’Mello, Ibrahim Ilahi, Kunwar Karim, Mubariz Maqsood, Adnan Moinuddin, Nardien Sedhom, and Julia Zhu in collaboration with the McMaster Demystifying Research Program.
We would like to also acknowledge the contributions of Dr. Takuma Isshiki, who conducted the wet transfer and provided guidance in the process. Additionally, we would like to also acknowledge Anmar Ayoub, who aided with materials and resources for the wet transfer. Finally, we would like to also thank Dr. Kjetil Ask and the Ask Lab for the additional guidance, insights and support.
Episode #1: Introduction to Western Blotting: Introduction to the Western Blot
Episode #2: SDS-PAGE: Western Blot: Introduction to SDS-PAGE
Episode #4: Electroblotting: Western Blot: Introduction to Electro...
Episode #5: Blocking: Western Blot: Introduction to Blocking
Episode #6: Immunodetection: Western Blot: Introduction to Immunod...
Episode #7: Results & Analysis: Western Blot: Result Interpretation
This video is for general and educational purposes only.
Subscribe to the Demystifying Research McMaster channel.
Copyright McMaster University 2022
#DemystifyingResearch #howtodoawesternblot #WesternBlot #howtodoawettransfer #wettransfer #immunoblotting #electrotransfer
References:
A good sandwich must be tight - change your blotting pads. (2016, May 17). Advansta Inc. https://advansta.com/good-sandwich-mu...
Arslan, J. (2019, December 2). Wet or dry protein transfer: Which one to choose for your western blotting? Novusbio.Com. https://www.novusbio.com/antibody-new...
Bass, J. J., Wilkinson, D. J., Rankin, D., Phillips, B. E., Szewczyk, N. J., Smith, K., & Atherton, P. J. (2017). An overview of technical considerations for Western blotting applications to physiological research. Scandinavian Journal of Medicine & Science in Sports, 27(1), 4–25. https://doi.org/10.1111/sms.12702
Bio-Rad. (n.d.). Western blot protocol. Bio-Rad. Retrieved April 23, 2022, from https://www.bio-rad-antibodies.com/we...
Goldman, A., Harper, S., & Speicher, D. W. (2016). Detection of proteins on blot membranes. Et al [Current Protocols in Protein Science], 86(1), 10.8.1-10.8.11. https://doi.org/10.1002/cpps.15
Kurien, B. T., & Scofield, R. H. (2015). Western blotting: an introduction. Methods in Molecular Biology (Clifton, N.J.), 1312, 17–30. https://doi.org/10.1007/978-1-4939-26...
Mahmood, T., & Yang, P.-C. (2012). Western blot: technique, theory, and trouble shooting. North American Journal of Medical Sciences, 4(9), 429–434. https://doi.org/10.4103/1947-2714.100998
Pettegrew, C. J., Jayini, R., & Islam, M. R. (2009). Transfer buffer containing methanol can be reused multiple times in protein electrotransfer. Journal of Biomolecular Techniques: JBT, 20(2), 93–95.
Protein Transfer Methods for Western Blot. (n.d.). Sinobiological.Com. Retrieved April 23, 2022, from https://www.sinobiological.com/catego...
The Protein Man. (2014, November 12). PVDF or Nitrocellulose - Which Membrane is Best? Gbiosciences.Com. https://info.gbiosciences.com/blog/bi...
Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76(9), 4350–4354. https://doi.org/10.1073/pnas.76.9.4350
Western blot transfer methods - US. (n.d.). Retrieved April 23, 2022, from https://www.thermofisher.com/ca/en/ho...
The video lists the materials and reagents needed for a wet transfer and how to conduct it. Note that there are several different kinds of transfer buffers used. One of the most common is the Towbin transfer buffer, named after one of the scientists who helped develop the western blot. It contains 25mM of Tris, 19.2 mM of glycine and 20% methanol.
This video was created by Kieran Anderson, Kevin Cao, Aniqa Chaudhry, Brooke D’Mello, Ibrahim Ilahi, Kunwar Karim, Mubariz Maqsood, Adnan Moinuddin, Nardien Sedhom, and Julia Zhu in collaboration with the McMaster Demystifying Research Program.
We would like to also acknowledge the contributions of Dr. Takuma Isshiki, who conducted the wet transfer and provided guidance in the process. Additionally, we would like to also acknowledge Anmar Ayoub, who aided with materials and resources for the wet transfer. Finally, we would like to also thank Dr. Kjetil Ask and the Ask Lab for the additional guidance, insights and support.
Episode #1: Introduction to Western Blotting: Introduction to the Western Blot
Episode #2: SDS-PAGE: Western Blot: Introduction to SDS-PAGE
Episode #4: Electroblotting: Western Blot: Introduction to Electro...
Episode #5: Blocking: Western Blot: Introduction to Blocking
Episode #6: Immunodetection: Western Blot: Introduction to Immunod...
Episode #7: Results & Analysis: Western Blot: Result Interpretation
This video is for general and educational purposes only.
Subscribe to the Demystifying Research McMaster channel.
Copyright McMaster University 2022
#DemystifyingResearch #howtodoawesternblot #WesternBlot #howtodoawettransfer #wettransfer #immunoblotting #electrotransfer
References:
A good sandwich must be tight - change your blotting pads. (2016, May 17). Advansta Inc. https://advansta.com/good-sandwich-mu...
Arslan, J. (2019, December 2). Wet or dry protein transfer: Which one to choose for your western blotting? Novusbio.Com. https://www.novusbio.com/antibody-new...
Bass, J. J., Wilkinson, D. J., Rankin, D., Phillips, B. E., Szewczyk, N. J., Smith, K., & Atherton, P. J. (2017). An overview of technical considerations for Western blotting applications to physiological research. Scandinavian Journal of Medicine & Science in Sports, 27(1), 4–25. https://doi.org/10.1111/sms.12702
Bio-Rad. (n.d.). Western blot protocol. Bio-Rad. Retrieved April 23, 2022, from https://www.bio-rad-antibodies.com/we...
Goldman, A., Harper, S., & Speicher, D. W. (2016). Detection of proteins on blot membranes. Et al [Current Protocols in Protein Science], 86(1), 10.8.1-10.8.11. https://doi.org/10.1002/cpps.15
Kurien, B. T., & Scofield, R. H. (2015). Western blotting: an introduction. Methods in Molecular Biology (Clifton, N.J.), 1312, 17–30. https://doi.org/10.1007/978-1-4939-26...
Mahmood, T., & Yang, P.-C. (2012). Western blot: technique, theory, and trouble shooting. North American Journal of Medical Sciences, 4(9), 429–434. https://doi.org/10.4103/1947-2714.100998
Pettegrew, C. J., Jayini, R., & Islam, M. R. (2009). Transfer buffer containing methanol can be reused multiple times in protein electrotransfer. Journal of Biomolecular Techniques: JBT, 20(2), 93–95.
Protein Transfer Methods for Western Blot. (n.d.). Sinobiological.Com. Retrieved April 23, 2022, from https://www.sinobiological.com/catego...
The Protein Man. (2014, November 12). PVDF or Nitrocellulose - Which Membrane is Best? Gbiosciences.Com. https://info.gbiosciences.com/blog/bi...
Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76(9), 4350–4354. https://doi.org/10.1073/pnas.76.9.4350
Western blot transfer methods - US. (n.d.). Retrieved April 23, 2022, from https://www.thermofisher.com/ca/en/ho...
2 سال پیش
در تاریخ 1401/06/09 منتشر شده
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