My gene belongs to Actinomycetaceae family and I want to do heterologous expression that gene in E.coli. I did this work from 4 months ago, but till now, I could not get any positive results. Do you have any suggestions for my science experiment?
i used pET28a(+) and expression host is E.coli BL21.
my gene size just 825bp. its around 30.3kDa
i tried with 1M IPTG (30microliter per 50ml of culture broth) at 20 C, for 18hrs. i had already sequenced my gene and its right
and one problem is running the SDS-PAGE, my protein size is 13.6kDa. So do you guys have any idea to see that band, because when i tried to run the Gel, i just can see band above 20kDa :((
I have never ever try western blot, coz till now i dont know yet my gene expressed or not :(((
Hi,
Are you absolutely sure you are using a BL21 (DE3) strain not just BL21? It is necessary for pET plasmids to work properly.
13.6 (or around 30.3?) kDa is absolutely OK for SDS-PAGE.
Dont bother with a gradient gel it is not necessary.
Use 14% or so tris-glycin (for a 13kDa protein preferably tris-tricin) SDS-PAGE
Rosetta cells are OK (remember to use a DE3 strain) but the tRNAs are on a plasmid so you need to use another antibiotics (Cam) which can slow down the growth.
Hi, Michal
what is so important of DE3? and Rosetta cells should use with Cm antibiotic?
its so difficult to change the yield of soluble form :(
for details on rosetta and RIL/RP see
http://wolfson.huji.ac.il/expression/rosetta.pdf
for DE3 see the following links
http://chem.winthrop.edu/faculty/grossoehme/link_to_webpages/courses/chem525/DE3.pdf
https://www.researchgate.net/post/What_is_the_difference_between_bl21_and_bl21DE32
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