Western Blotting Woes

For the past few weeks, I’ve been working diligently to try and prepare lysates for my experiments. With most of my lysates finished, I prepared to move into western blotting for visualization. At the NCI, I performed many western blots, so I was fairly confident in my ability to get good bands. During the school year, I also tried to visualize some proteins, to no avail. I was hopeful to get better results over the summer, but we are still trying to troubleshoot. We’ve been trying to determine if the antibodies are working properly, and to see whether or not the transfer was good. Using a stain called ponceau, we found that our first transfer method was not actually transferring the protein onto our membrane. Because of this, we switched to a semi-dry transfer method. We think that we have figured out what the problem is, so this week we are looking forward to getting nice bands on our film!

Comments

  1. tmprioleau says:

    I hope that your western blots work out in the coming weeks! I am exciting to see the results of your experiment.

  2. amzhang says:

    Oh no! I am so sorry this is happening to you! Maybe try remaking the running buffer? Do you put your secondary antibody in milk? Thats so crazy since you have experience, it’s really unheard of.

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