| | |||||||
| Register | Search | Today's Posts | Mark Forums Read |
| Protein Forum Protein Forum |
| | LinkBack | Thread Tools | Display Modes |
|
#1
| |||||||||||
| |||||||||||
| am trying to express an 18kDa protein which I had synthesized and codon optimized for ecoli. It has been successfully inserted into pET21a(+) (confirmed via sequencing and restriction digest) but growth in BL21(DE3) cells has been less than optimal. I am following the protocol we have established in our group for this same protein from different species (mouse, bovine, trout...while this new one is zebrafish): 4 hours growth in LB media with AMP at 37 degrees (OD at least 0.6), induction with 1mM IPTG, grow for another 4 hours at 37 degrees. The SDS PAGE gel shows my protein is there, just not over-expressing (I am going to need a lot - and even after purification it is still just a small band). I have been playing with IPTG concentration (0.5-2.0 mM does not seem to change), induction time (4-6 hours after induction does not change anything), and temperature (O/N induction at 24degrees didn't work). Any suggestions would be great. |
| Tags |
| expression , low , pet21 |
| Thread Tools | |
| Display Modes | |
|
|
| | ||||
| Thread | Thread Starter | Forum | Replies | Last Post |
| Human Cytome Project - Update 24 Jan. 2005 | Peter Van Osta | Cell Biology and Cell Culture | 1 | 08-01-2010 02:18 PM |
| Human Cytome Project - an idea - Update 19 April 2005 | Peter Van Osta | Cell Biology and Cell Culture | 1 | 06-01-2009 02:17 PM |
| qPCR NEWSLETTER - October 2006 | Editor www.Gene-Quantification.info | Protocols and Methods Forum | 0 | 10-26-2006 12:25 PM |
| A Human Cytome Project - an idea - Update 14 March 2005 | Peter Van Osta | Cell Biology and Cell Culture | 0 | 03-14-2005 01:27 PM |
| Human Cytome Project - Update 6 Jan. 2005 | Peter Van Osta | Cell Biology and Cell Culture | 0 | 01-06-2005 10:18 AM |