Re: [ccp4bb] How to express a 95KD FAD protein

[Kn Ly]

try codon optimisation
use Rosetta 2 strains
fuse to MBP
I expressed a yeast protein of ~100 KDa, expression was low until I fused it
to MBp (resulting protein ~ 150 KDa)

Re: [ccp4bb] Purification

[Kn Ly]

Hello everyone,

Just want to say thanks for your great ideas and time to reply my question.
Hope I will solve my problem soon

Kien

[ccp4bb] purification

[Kn Ly]

Hello everyone,

I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein
is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa.

However, the protein get severely degraded so after putting through a Ni-NTA
column, the protein came out with a lot of contaminant bands. I did a
western blot using antibody against his tag. The total cell lysate gave
signals in many bands. The flow through did not give any signal and the
eluted fraction again gave many band signals, indicating the protein got
degraded copiously even before purification.

I used Roche protease inhibitor tablet and still got a lot of degradation.
Can anyone suggest a way to avoid the problem or a purification method so
that I can purify the intact protein while keeping away the unwanted
degraded fractions.

Thanks heaps in advance.

Kien

[ccp4bb] native gel

[KN]

Dear all,

I  am wondering if anyone is working on Blue Natve PAGE or other alternate
methods. I need some help to troubleshoot my Native PAGE experiments. It
will be great if anyone can help me in this regard. I am working on membrane
proteins which have pI around less than 7. i need to run Native PAGE for my
samples which are purified with nonionic detergents. When i tried to run
inhouse 4-16% gradient Native gel at running buffer pH of 8.9, the samples
are getting stuck at the wells itself. I just found out that invitrogen Blue
Natvie PAGE may be a good option but due to the cost if anyone can suggest
me some other alternate methods, I will really appreciate it.

Thanks in advance,

regards,

Jothi,

[ccp4bb] LDAO SDS-PAGE

[Subscribe Ccp4Bb Kn L]

Hello everyone...

I have a protein solubilised in 1% LDAO, 50 mM Phosphate pH 7.5, 150 mM
NaCl. When I mix it with laemmli buffer and heat at 95C, 3 mins for
SDS-PAGE, it aggregates (as in when you mix GuHCl with SDS) and can't be
loaded onto the gel.

Does anyone know why it happens? Thanks a lot!!!

Kien