Getting Good Bands

MVIMG_20190722_142925_2

Last week, I finally got my first working blot of the summer! After some trial and error, we realized that we should remake our running buffer for our protein gels. Making this was a long process, especially because I had to wear a mask for the whole time due to the toxicity of one of the components. Additionally, pH-ing the buffer meant that I had to wear the mask for longer, which made it an overall unpleasant process. After having a fiasco with making gels, I was finally able to run my lysate and get the blot started. I learned how to do a new method of transferring, the wet transfer, which was interesting, but takes much longer than some of the other methods. We also added milk to our secondary antibody, and increased the concentration of the antibody. While waiting for the film in the developer, I was quite nervous, but ecstatic when the film came out with clean, crisp bands. This was what I have been waiting for, and I’m excited to get to visualize the rest of my work from this summer!

Comments

  1. kmreed01 says:

    Hi Emma! I’m so glad you were able to finally get a working Western blot! They can certainly be frustrating to troubleshoot. How does the wet transfer method differ from other ways of transferring the proteins from the gel to the membrane?

  2. ktkelley says:

    Wow! Emma I’m really glad that you were able to obtain a good blot and that you have managed to find a system that works. While it does sound like more of a hassle than usual, it must be a great feeling to have a method that was able to get you such great looking bands. I hope that you find this to work consistently and that you see many great blots in the near future.

  3. njlignore says:

    Hi Emma,
    It is so exciting that you were able to troubleshoot the issues you were experience with your gel. I am also happy to hear that you learned a new method of transferring and I am sorry to hear it takes so long. Perhaps there is a condensed version you can use as your results become more consistent? I hope you continue to get good results for the remainder of your project. Having to start from the beginning on any project can be challenging and I myself have found myself starting from scratch at several points in the summer. What specifically led you to realize you had to remake your running gel? I noticed that you increased the amount of anti body present in your successful run in addition to adding milk to your second antibody. Which do you think had more of an affect on your results?

    Best,
    Niko

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