Good morning/afternoon
I am over-expressing a bacterial protein (dimer) in BL21 E. coli, which it works even with 0.04mM of IPTG, incubated at 26C. However, when I want to check the solubility of my protein I see so many bands, even in the right molecular weight size, and I get confuse.
To check the solubility I used Bugbuster. I spin 1ml of induced cells, add 300ul of bugbuster to the pellet, and 20uL of EDTA free-protease inhibitor cocktail, with a 20 min slow mixing at room temperature. Then centrifugation at 16000xg for 20 min at 4C. I use 10uL of supernatant for SDS-PAGE analysis.
I have many question like:
*is it possible that my protein is soluble but I get only a fraction of it, by using bugbuster protocol? since I use 10uL for the SDS-PAGE analysis, maybe it is diluted and I can not see the big band, as in the case when I check the expression using whole cells.
*I can even over express my protein using 0.04mM of IPTG at 26C incubation for 6H. Butif it is insoluble after all, should I try lower IPTG concentration and temperature to try to make it soluble?
Please find attached the SDS-PAGE before and after induction using the whole cells, and the supernatant after the bugbuster treatment. My protein is a dimer, around 103 KDa and 28KDa, the larger and shorter fraction, respectively.
Sorry for any English misspelling. And also, this is my first time working with protein heterologous expression, so I get so many questions. Sorry if my questions sound stupid.
And Thanks for any help or guide you can provide me.
Do you have an antibody against the proteins you are expressing? Whilst it looks like you are getting expression of your proteins it would be best to assume nothing and check for expression via western blot. Assuming that the strong bands in your whole cells lysate ARE the proteins of interest then yes it does look like the majority of it is insoluable. Again a western blot would indicate whether any of your expressed protein is soluble.
............actually having just performed a quick search there are already posts on this site with helpful suggestions
https://www.researchgate.net/post/How_much_of_IPTG_concentration_is_used_for_induction_of_gene_in_Ecoli
Could you check the pellet with SDS-PAGE? This information will help to judge the solubility of your recombinant proteins in E. coli. Another thing you need to pay attention is the Bugbuster is not suitable for all proteins, and you need to know more about your recombinant proteins and then you can judge.
Make 15-20% (w/v) cell suspension in appropriate buffer, sonicate and centrifuge at 25000 g. Collect supernatant (soluble) and redissolve pellet (insoluble proteins). Load supernatant and pellet fraction on PAGE and see where the protein is present.
You can try different methods for break the cells or change the reagent for the exaction (I use the cell lytic from Sigma).
Also for me the sonication is the best way !!! eheh
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