Dear all,
I have used the following buffers for purification and dialysis. this is
fyi.
*Lysis buffer*:
25mM Tris pH 7 or 7.5 or 8
100-500mM NaCl (increase in salt concentration increased precipitation of
the protein in the column itself)
*5mM Beta mercaptoethanol *
*0.5% Triton X 100 *
I have tried with other buffers also.
a. HEPES buffer pH7.5
b. Phosphate buffer pH 7.8
c. MOPS buffer pH 8
*Wash and Elution Buffer*:
25mM Tris pH 7 or 7.5 or 8
100-500mM NaCl
20 and 30mM Imidazole for wash
300mM for elution
*Dialysis Buffer*:
1. Tris 25mM pH 7
2. Tris 25mM pH 7.5
3. Tris 25mM pH 8
4. Tris 25mM pH 7.5, 5% glycerol
5. Tris 25mM pH 7.5, 10% glycerol
6. Tris 25mM pH 7.5, 20% glycerol
7. Tris 25mM pH7.5, 50mM NaCl
8. Tris 25mM pH7.5, 100mM NaCl
9. Tris 25mM pH7.5, 1mM MgCl2
10. Tris 25mM pH7.5, 50mM NaCl, 50mM L-Arg, 50mM L-Glu
In all these cases the protein precipitates. i have tried to do buffer
exchange also. i can see precipitate sticking on the walls of the tube
during the process.
On Thu, Mar 30, 2017 at 10:14 AM, Debanu Das <debanu@gmail.com> wrote:
> Hi Akila,
>
> In addition to what others have asked about the dialysis buffer, a few
> more comments that might help to decide next steps because the
> precipitation (note precipitation and aggregation are related but not
> synonymous) may be due to several different or related reasons:
>
> 1) At what stage are you dialyzing? Is it after SEC? Could your
> protein be too concentrated at that point since your yield is high
> leading to some precipitation? How severe is the loss?
> 2) Did you try more purification before dialysis?
> 3) Are you removing the detergent in the buffer you are dialyzing against?
> 4) Can you try buffer exchange during concentration instead of dialysis?
> 5) Try increasing your NaCl concentration and adding 5-10% glycerol to
> improve protein solubility.
> 6) Did you try cleaving the C-term His-tag before dialysis? Did you
> try N-term His tag?
> 7) Do you really need to dialyze? Did you try assays or
> crystallization trials with the purification buffer? You can run a SEC
> column without imidazole to remove that before crystallization.
> 8) PSI/SBKB TargetTrack can be great resource to look at
> expression/purification protocols for similar proteins:
> http://sbkb.org/tt/
> 9) Also look up the Tb Structural Genomics resource to see if there is
> anything on this target or related targets: http://www.webtb.org/
>
> Best,
> Debanu
>
> On Wed, Mar 29, 2017 at 6:38 AM, Akilandeswari Gopalan
> <akilaibt2...@gmail.com> wrote:
> > Dear all,
> > I am a PhD student doing structural studies on a few proteins from
> > Mycobacterium tuberculosis. The gene encoding the proteins I work on are
> > cloned into pet22b with c terminal His tag. the proteins are expressing
> > well. upon purification I am getting good yield of protein but during
> > dialysis, the proteins precipitate. Kindly suggest some solutions to
> avoid
> > aggregation. pI of one protein is 9.7 and that of the other is 5.6
> > I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM
> > beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer
> with
> > 20-30mM imidazole for washing and 300mM imidazole for eluting the
> proteins.
> >
> > Thank you
> > Regards
> > Akila
> >
> > --
> > Akilandeswari G
> >
>
--
Akilandeswari G
JRF
C/O Dr. Alamelu Raja
National Institute for Research in Tuberculosis
Chetput, Chennai
