Western Blot Analysis with Rat Liver Homogenate

Western blot analyses are run to separate and identify proteins. First, the proteins are separated through gel electrophoresis. The proteins are transferred to a membrane where they are marked with antibody, and a film is developed for visualization (‘Western Blot: Technique’).

Gel preparation:

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Waiting for the gel to solidify:

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Adding the samples of Rat liver homogenate into the gel:

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Waiting for the gel to run:

At the beginning:

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Almost done:

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Here, the protein is being pushed through the gel by an electric field. The purple dye is used to identify the edges.

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The next step is blotting, where the proteins on the gel are transferred to membrane paper using a semi-dry cell.

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After waiting for an hour and a half, the membrane is stored in the cold room overnight at 4 degrees Celsius. The membrane can also be stored at room temperature for an hour.

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In the morning, I cut the membrane in half and pour two different antibodies onto each.

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These are antibodies oatp1 and ae745.

I placed them on the orbital shaker for two hours.

I proceeded by washing away non-specific proteins!

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I run antibody 2 with Rabbit HRP and performed a second round of washing.

Lastly, I develope films of the western blot analysis in the film room.

Here is the final result!

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The black lines are where the antibody bound to the protein. Since they are clearly visible, this western blot analysis was a success!

I would like to give a special thanks to Peggy Wang for mentoring me throughout this process, and to Dr. Allan Wolkoff for providing the opportunity to work in his lab!

Works Cited:

Mahmood, T., & Yang, P. (2012, September). Western Blot: Technique, Theory, and Trouble Shooting. Retrieved July 21, 2017, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/

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