Look to your lysis pellet and supernatant to tell if your lysis strategy worked well!

Описание: Look to your lysis pellet and supernatant to tell if your lysis strategy worked well! (better hope so or your protein is still trapped inside the cell! and therefore pellets out…)

If your lysis went well, you should get a small, tight pellet, often with multicolored rings and a shiny edge ring. And the supernatant (liquid) should be yellowy

If your lysis didn’t go well, you will have a bigger, softer, pellet without those rings, and the supernatant will be super dilute & barely yellow (note: I labeled the picture “clear” but I meant “colorless,” sorry!)

To help ensure that your lysis went well before you spin things down, look at the lysate over the lysis process (e.g. in between sonication cycles) - you should see it go from a thick milky look to more of a skim milk or dirty water look. It won’t get clear, but it will clear up a bit. It’s sometimes easiest to see if you hold it up to the light.

Over time, you’ll get a sense of what to look for so that you lyse thoroughly, but not excessively. Too excessive sonication, etc. can cause the protein to denature (unfold) and stuff which is bad…

But too gentle of lysis and your protein will end up in the pellet (which you want to only contain membrane bits and insoluble stuff, not intact cells) - and then you can’t even tell if your protein was soluble or not.

Hope that helps - it’s a lot of trial, error, and optimization, especially since, as I’ve discovered, sonicator settings differ for different sonicators and are sometimes quite finicky!

More on lysis methods: blog form: https://bit.ly/homogenization ; YouTube:    • Homogenization and lysis (cellular disrupt...    

more on sonication: https://bit.ly/ultrasonicshearing ;    • Ultrasonication (often just called "sonica...    

more on troubleshooting if your expression isn’t going well: http://bit.ly/wherestheprotein

lots more on protein purification: http://bit.ly/proteinpurificationtech &    • Protein chromatography     
           
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com