Expression and Purification: Tips and Tricks: Difference between revisions

Expression and Purification: Tips and Tricks (edit)

707 bytes added , 10 February 2008

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 In practice, it is a good idea to take your protein after the first purification step (such as metal affinity column if you have his-tag) and give it a go with crystallization screens.  Protein crystals often take two-three weeks to appear and you can use this time to optimize your purification protocol.  Of course, it is important to realize that in some situations only final purified product will crystallize, so if preliminary trials with "not-so-pure" protein failed, you should consider running the same screens second time with highly purified batch.
+== Too little protein to see in the gel ? ==
+Try TCA precipitation - an old time classic!
+===Materials required===
+*  100% (w/v) Trichloroacetic acid (dissolve 50g TCA into ~35mL water)
+*  Acetone
+===Procedure===
+*  1. Add 1 volume TCA to 4 volumes of protein sample.
+*  2. Incubate on ice for 10 minutes.
+*  3. Spin at 14K rpm for 5 minutes.
+*  4. Remove supernatant, leaving protein pellet intact.
+*  5. Wash pellet with 200µL ice-cold acetone.
+*  6. Spin at 14K rpm for 5 minutes and discard supernatant.
+*  7. Repeat steps 4-6 for a total of 2 washes.
+*  8. Dry pellet on a heat block at 95°C for 5-10 minutes to drive off acetone.
+*  9. Add loading buffer to sample and boil before loading on gel.