Hello Rafael No problem, thank you for confirming.
Best wishes Jon Cooper -------- Original Message -------- On 10 Jul 2020, 14:27, Rafael Marques wrote: > Thank you for pointing me this out. I don’t know why I said BL21...maybe in > the rush to say to use Rosetta for protein expression. We use Dh5a for > plasmid replication (although I have already used BL21 for plasmid > purification and it worked nicely). My bad. > > Rafael Marques da Silva > > Mestrando em Física Biomolecular > > Universidade de São Paulo > > Bacharel em Ciências Biológicas > > Universidade Federal de São Carlos > > phone: +55 16 99766-0021 > > "A sorte acompanha uma mente bem treinada" > ________________________________________________ > > De: bogba...@yahoo.co.uk > Enviado:sexta-feira, 10 de julho de 2020 00:30 > Para: [Rafael Marques](mailto:rafael_mmsi...@hotmail.com) > Cc:CCP4BB@JISCMAIL.AC.UK > Assunto: Re: [ccp4bb] RES: [ccp4bb] Looking for suggestions with protein > expression > > Re: "In our lab we generally use BL21 for plasmid replication." > > I am interested because on the occasions that I prepared plasmids in BL21 > (usually by mistake), they were never any good for cloning or sequencing. I > always had to transform them back into a cloning strain and do another > plasmid prep! Probably my incompetence! > > Jon Cooper > > On 9 Jul 2020 21:44, Rafael Marques <rafael_mmsi...@hotmail.com> wrote: > >> Hi Umar, >> >> I must say that it would be better use as an expression system Rosetta DE3 >> or Rosetta-gami instead of BL21. In our lab we generally use BL21 for >> plasmid replication. >> >> Also, there are a few things that you could try before trying another >> construction. >> >> - Lower the temperature during the expression. >> - Try to use a different range of pH in your buffer. Maybe you could add a >> bit of NaCl (150 – 300 mM) and/or glycerol (2 – 10%) >> - I must say that I have already obtained very different results using Co >> or Ni columns for IMAC. You could take a look at this. >> >> Regards >> >> ______________________________________________________ >> >> Rafael Marques da Silva >> >> Mestrando em Física Biomolecular >> >> Universidade de São Paulo >> >> Bacharel em Ciências Biológicas >> >> Universidade Federal de São Carlos >> >> phone: +55 16 99766-0021 >> >> "A sorte acompanha uma mente bem treinada" >> >> ________________________________________________ >> >> De: [Lau Kelvin](mailto:kelvin....@epfl.ch) >> Enviado:quarta-feira, 8 de julho de 2020 16:22 >> Para: CCP4BB@JISCMAIL.AC.UK >> Assunto: Re: [ccp4bb] Looking for suggestions with protein expression >> >> Hello Umar, >> >> I would not pin down your difficulties solely due to an Fe-S proteins. I >> have produced some with no fusion partners and they work wonderfully. They >> were expressed in an aerobic environment and then reduced in an anaerobic >> one before usage in reactions. >> >> 1) On the Fe-S side, there are plasmids you can co-transform to increase >> Fe-S production. This plasmid pH151 has the synthetic genes necessary for >> Fe-S formation. >> >> https://www.jbc.org/content/279/33/34721.abstract >> >> 2) On the general protein side, have you hhpred your protein? Different >> constructs (not just tags), temperature? Strain? Media? >> >> 3) For these proteins we typically use His Excel (or Protein Ark Ni2+ >> Advance) that is resistant to most chelators since more often than not, they >> contain other metals and can snatch Ni2+ from normal Ni-NTA resins. Strep >> tags also work well. >> >>> On Jun 27, 2020, at 9:14 AM, Umar Farook <umarfaroo...@gmail.com> wrote: >>> >>> Dear All, >>> >>> Sorry for an offtopic question, your suggestions are highly appreciated. >>> >>> We have been working on iron sulfur cluster binding protein, which is >>> usually expressed as a nice soluble protein expressed in BL21 cells but >>> aggregated in the affinity column itself and unable to recover from it. We >>> had made n number of truncations and fused to soluble tags such as MBP, but >>> always ended up in large aggregates. Anyone has experience in working with >>> iron-sulfur cluster binding protein before, please let us know the critical >>> steps in purification of such proteins, whether you have completely done >>> the expression, purification and crystallization in anaerobic conditions? >>> or else changing the expression system to eukaryotic system such as Baculo >>> or HEK 293T would help? >>> >>> Please share your valuable experience, thank you. >>> >>> -- >>> >>> Best Regards, >>> >>> Umar Farook >>> >>> To unsubscribe from the CCP4BB list, click the following link: >>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > --------------------------------------------------------------- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
