I do agree with the others about what they said previously and I would not
bother about the ~60kDa protein. I suppose the tag is in the 15kDa one by the
amount of protein you have in the gel. I think you might be facing two
different “problems”. One of your proteins (or the complex) is precipitating
during SEC. Do you centrifuge your samples before running SEC? Do you keep them
in 4ºC? What concentration do you get after your first elution? Your sample may
look very clean in solution but maybe this is not really real.
The other “problem” is that maybe your proteins are forming bigger oligomers in
solution. You can try native gel to find this out but firstly I would use
another SEC column, a bigger one. Superdex 200 10/300 is an analytical column
and should not be used for purification itself (although I have done this many
times). I would try maybe S200 16/60.
Best
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: Dilip 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
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 while the other one is15 kDa. I do not think that
they are making that strong heterodimer. The total molecular of the complex
would be ~ 52 kDa but now it is around 60 kDa. I am currently using lower
temperature (16 ᵒC) but can try around 12 with low IPTG conc.
Regards
Dilip Badgujar
On Mon, Jul 12, 2021 at 11:56 PM zaigham khan
<mahmood.zaig...@gmail.com<mailto:mahmood.zaig...@gmail.com>> wrote:
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 buffer, and despite boiling
of the samples. Could this be that 60 KD is actually the hetero-dimer? One can
perform western blotting, followed by the use of anti-polyhistidine and
anti-Streptavidin antibodies on separate blots to confirm the suspected bands.
Likewise SEC followed by WB may confirm the identity of the eluted proteins in
different fractions after size exclusion chromatography. You may also cleave
the tags, and then see the magic!
Tom has correctly pointed out that induction at lower temperature is best
achieved upon incubation of culture so that the temperature is dropped before
the induction.
Bon Voyage!
-Z
Zaigham M Khan, PhD
Associate Scientist
Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York
United States
On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar
<dilip....@gmail.com<mailto:dilip....@gmail.com>> wrote:
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 during growth of pre-culture
and during induction. Initially, cells were grown at 37 °C till OD reaches to
0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When I do
purification using Streptactin resin; I can see proteins of my interest bound
to the resin along with contaminating protein at 60 kDa. I have tried
performing size exclusion as a follow up step but they are co-eluting in void
volume. I have also tried to wash with MgCL2-ATP solution but it co-elution
with contaminant. I am looking for valuable suggestions to avoid the
contamination during or after expression.
Thanks in advance.
Regards
Dilip
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
--
Dilip C. Badgujar, (PhD)
Post Doctoral Fellow,
IIT Bomaby
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/
