Lab tip: Check pre-& post-induction samples on an SDS-PAGE gel to see if your protein expressed

Before you try to purify a protein, check that it’s actually there to purify! Here’s how…

Take 1mL samples pre-induction & at time of harvesting

Pellet the cells out by centrifuging it at top speed for ~10min and removing liquid media.

Prepare SDS-PAGE Sample
add 50uL 1X sample loading buffer (this has a detergent so it will help small-scale lyse things)
heat 10-15min (can stick in PCR machine set to incubate at 95-100°C)
centrifuge briefly
load supernatant (~5uL)

Run gel

Look for the appearance of a new band at expected size

If you don't see it (especially if you were using a T7 system, where your protein should be majorly overexpressed (since it gets (all the copies of) a whole polymerase devoted to it!), what can you do?
* check your plasmid if you haven't recently - make sure it doesn't have any mutations (other than site-directed ones you might have made of course...)
* prepare fresh IPTG & try again
* check the gunky pellet part in case your protein is getting made, but is going into inclusion bodies
* try a different expression strain or at least a different aliquot or batch

If you see a faint band, try adjusting IPTG concentration and/or expression temperature and/or expression time

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

more on IPTG induction:  http://bit.ly/bacterialproteinoverexp... &  IPTG induction using the lac promoter
          
more about all sorts of things:  #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com