Hvvxbxhxxhhfjxjdjdhhdydfyhat the 60 kDa is not your biggest concern.
Optimize soluble protein expression and pay attention to cell lysis. Is
the sample remaining cold throughout the process? Are you using enough
volume of buffer relative to solid cell pellet?
>
> Also, from the gel it does not
p Badgujar<mailto:dilip@gmail.com>
Enviado:terça-feira, 13 de julho de 2021 11:23
Para: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Assunto: Re: [ccp4bb] 60 kDa contamination in Rosetta cells
Hello,
Please find the attached gel picture for the reference and some additional
inform
board on behalf of Tom Huxford
Sent: Tuesday, July 13, 2021 11:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells
I agree with Dhiraj' suggestion that the 60 kDa is not your biggest concern.
Optimize soluble protein expression and pay
I agree with Dhiraj' suggestion that the 60 kDa is not your biggest concern.
Optimize soluble protein expression and pay attention to cell lysis. Is the
sample remaining cold throughout the process? Are you using enough volume of
buffer relative to solid cell pellet?
Also, from the gel it
: [ccp4bb] 60 kDa contamination in Rosetta cells
Hello,
Please find the attached gel picture for the reference and some additional
information.
Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease inhibitor
cocktail.
SEC column – Superdex 200. 16/300
One of the proteins is 37 kDa
Hey Dilip,
There are many reasons for this observation. Rafael is right, please do
share the image of the gel. Also what are the exact sizes of the two
proteins that you co-expressed? I have observed the heterodimeric and
pentameric proteins on SDS-PAGE albeit the presence of DTT in the sample
That is almost certainly GroEL. I would start by lowering expression
temperature. Maybe try moving your warm cultures to room temperature (we use
stir plates) as soon as you begin to see growth. Allow them a couple of hours
to cool and then induce with low IPTG (0.1 mM) for 14-16 hours
sorte acompanha uma mente bem treinada"
De: Dilip Badgujar<mailto:dilip@gmail.com>
Enviado:segunda-feira, 12 de julho de 2021 03:45
Para: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Assunto: [ccp4bb] 60 kDa contamination in Ro
Greetings, everyone
I am trying to co-express two mammalian proteins (less than 50 kDa MW) in
Rosetta cells but getting a contaminating band around 60 kDa. One of the
construct is in pET28a with His tag while the other is in pET21c with Strep
tag and I am adding all the three selection markers
