Troubleshooting protein expression

When you go to purify the protein (assuming you’re purifying a non-secreted, non-membrane protein (i.e. a soluble cytoplasmic protein)), you break open the cells (lyse them), spin that lysate super fast in a centrifuge to pellet out the insoluble stuff (membrane bits, etc.), and then purify the protein of interest from the liquid part (supernatant) containing the soluble things. If you’re trying to purify a protein and you “can’t find it” there are a few possible culprits 
- the protein wasn’t expressed (the mRNA didn’t get translated) 
- the protein expressed but not “properly” so it’s hidden with the insoluble membrane gunk you pelleted out 
- the protein was expressed but then degraded 
- your ID method is flawed (problem with the purification if that’s how you’re checking or antibodies if you’re checking via western blot) 

Sometimes, of course you have a combination of these things, especially because if they’re expressed “badly” they often get degraded.  

Sometimes, especially when you try expressing a non-bacterial protein in bacteria, your protein folds incorrectly and it ends up in blobs of insoluble gunk called inclusion bodies. These inclusion bodies consist mainly of aggregated (clumped-together misfolded protein). This can especially tend to happen when the cells make too much of the protein too quickly, so you might be able to avoid them forming altogether by using less “aggressive” expression techniques as I’ll get into more later in the post (e.g. lower the temp, express for a shorter period, induce with less IPTG). 

I will go into some tips and tricks for things you can do to try to fix what’s wrong - but it’s important to first know what’s wrong to need to fix!