Each protein’s different, so the process has to get tweaked & optimized for different proteins. And for your goals - how pure do you need it to be? And resource availability - do you have access to an AKTA But here, I’m just going to take you through the process of a scientist producing and expressing, purifying, a protein and studying it with structural biology techniques like x-ray crystallography & cryo-EM
note: I made this as a pre-class lecture for one of my biochem classes to help them prepare for a jigsaw paper discussion activity I'm planning to do
page of protein purification posts: bit.ly/proteinp...
blog form with links to more on each of these things - bit.ly/proteinc... RU-vid: • Overview of a "typical...
We can break up the workflow into 3 main parts:
- MOLECULAR CLONING - this is where we take the gene* (DNA instructions for making the protein) from its original home and put it into a VECTOR (a manipulatable piece of DNA that can serve as a “vehicle” for getting those instructions into cells).
- EXPRESSION - this is where we have the bacteria make the protein for us (we can also express trickier proteins in insect cells or mammalian cells - I express most of my proteins in insect cells, but bacteria are simpler and simpler to explain!)
- PURIFICATION - this is where we break open the cells & isolate the protein we want from all the other stuff. It usually involves several types of PROTEIN CHROMATOGRAPHY, where we flow our sample through columns filled with special little beads (resin) that separate proteins based on how their different properties (charge, size, etc.) influence how they interact with those beads
Each of these main parts has multiple sub-parts. Check out the blog link for links to more!
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0
24 сен 2024
