I extracted my brain organoid samples recently and ran them on SDS-PAGE. These brain organoid samples were coated with Matrigel, I tried to remove as many as I can by washing them in cold 1X PBS but definitely, there are some leftovers. Then I processed these in lysis buffer supplemented with 8M urea and boiled them at 95C before running them on 10% acrylamide gel. However, these samples ran funny and the bands look smeared (as shown in pictures). Can anyone advise on this matter and how can I fix this problem?
Thank you in advance for your suggestions.
Hello Yen Teng Tai
Your ladder has been resolved properly, so definitely your samples have some issues. It is definitely not related to the voltage/current or your gel composition/preparation. You need to alter your lysis buffer. Such smeary patterns are usually obtained when your protein samples have a high concentration of salts or co-solutes. Another possibility is that you made loading too much of the total cell lysate (with large amounts of protein) leading to this. Try reducing the concentration of salts and don't load more than 20-30 ug of your protein on the gel. It may just work for you. Good luck!
Use new solutions and buffers, make sure to adjust the concentration of salts in the solutions and buffers (no more than 1x), and the pH must be controlled.
Also, make sure that the current was high or there was heat in the tank.
https://www.researchgate.net/post/What_is_the_reason_for_smile_bands_in_my_blot
https://www.researchgate.net/post/What-could-cause-these-frowning-bands-seen-in-electrophoresis-SDS-PAGE
https://www.bosterbio.com/protocol-and-troubleshooting/western-blot-troubleshooting
http://www.protocol-online.org/biology-forums-2/posts/28568.html
Hello Yen Teng Tai
Since your ladder migrated ok, the problem is not the current but it could be a problem in the preparation of lysis buffer, sometimes an excess of salts could lead to such smears. Try as well centrifugate your samples at maximum speed for 15'' before loading into the wells, this can also help to remove any precipitates that could trigger smearing in your gels. Good luck ;)