Hello, Dilip.
You have not told us about your buffer composition. Also, it would be great if
you could provide us a pic of your gels. I would suggest to increase the salt
concentration (~300 to 500 mM NaCl) to check if by doing this the interaction
between contaminant and your samples stops. Are your proteins monomers in
solution? If you are not sure, try a bigger SEC column.
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: Dilip Badgujar<mailto:[email protected]>
Enviado:segunda-feira, 12 de julho de 2021 03:45
Para: [email protected]<mailto:[email protected]>
Assunto: [ccp4bb] 60 kDa contamination in Rosetta cells
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
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