From: Jerry McCully
Date: 7 March 2012 14:50
----------
From: lieh low
Hi Jerry,
We have used 0.05% Triton X-100 in our wash buffer to wash the protein
on column (Ni-IMAC for His-tagged proteins and MabSelect for
antibodies). 20 column-volumes wash followed by 20 column-volume wash
without detergent is typically enough to reduce the endotoxin
significantly.
I would also suggest checking your FPLC system, and decontaminate with
2N NaOH if neccessary.
Hope that helps
Regards,
ray
----------
From: Kevin Jin
I tried to use 250mM Tris pH 7.6, 1M NaCl and 5~10% 2-propanol for
dialysis. It works,
----------
From: Taylor, Jonathan D
Dear Jerry,
I've just become aware of this spin column to remove endotoxin, marketed by Generon, UK.
http://generon.co.uk/index.php?route=product/category&path=238
Good luck,
Jon
________________________________________
----------
From: Pius Padayatti
This is in response to a comment to this thread
Kevin,
Could you explain how that worked?
How do you know your method worked?
Did you estimate the lipopolysaccharide before and after the method?
The method already mentioned here to wash using TritonX100 makes sense.
by washing bound protein sample May able to phase separate into
detergent micellar phase and get rid of endotoxins.
Most widely accepted method to separate is by two phase micellar system.
above the CMC endotoxins will be accomodated by micelles through non-polar
interactions of alkyl chains of lipidA.
Padayatti PS
----------
From: Kevin Jin
To Pius,
In my case, the protein/biopolymer is Lysine/amine riched.
1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
then provide positively charge to LPA. Of course, some metal cations
could also be involved in LPA binding.
2. Tris with the NH2 group could also be protonated and positively
charged under the same pH condition. This is why I use high
concentration Tris here.
3. Tris buffer could function as an ionic-exchange competitor for LPA
binding. If you look those commercial available Endotoxin assay kits,
they also use Tris buffer.
4. IPA could change the charge distribution on the surface of protein.
In usual, high% IPA could remove LPA efficiently but may cause protein
denature. In lot of cases, 5~10% IPA could help protein
crystallization to a larger size. As a tradeoff, it may also bring
down the packing quality.
In this case, I used it for endotoxin removal.
5. NaCl (you can try other salt) would provide additional ion-strength
to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.
6. If you can add EDTA to remove metal cation, it may be even better.
In my case, I could not use EDTA.
I tested the sample (1600EU/ml). After 3 days dialysis, it went down
to < 5EU/ml. The assay kit for LPA measurement was from Charles River
(?). The procedure is too long to present here. The good thing is that
your sample would not be dehydrated and refold again.
I am saying this will work for protein in all of case, but you try in
an eppendorf tube like amini-prep.
I guess this method may have been patented.
----------
From: Kevin Jin
Date: 7 March 2012 14:50
Dear All;
We purified a His tag protein by Ni-NTA and gel-filtration from E.coli.
We tried two endotoxin removal resins from Pierce. However, it is hard to remove the endotoxins in the purified protein because the protein bound to the resin as well.
This protein contains quite a few aromatic residues and has a pI around 6.
Any ideas to remove the endotoxins will be highly appreciated.
best regards,
Jerry McCully
We purified a His tag protein by Ni-NTA and gel-filtration from E.coli.
We tried two endotoxin removal resins from Pierce. However, it is hard to remove the endotoxins in the purified protein because the protein bound to the resin as well.
This protein contains quite a few aromatic residues and has a pI around 6.
Any ideas to remove the endotoxins will be highly appreciated.
best regards,
Jerry McCully
----------
From: lieh low
Hi Jerry,
We have used 0.05% Triton X-100 in our wash buffer to wash the protein
on column (Ni-IMAC for His-tagged proteins and MabSelect for
antibodies). 20 column-volumes wash followed by 20 column-volume wash
without detergent is typically enough to reduce the endotoxin
significantly.
I would also suggest checking your FPLC system, and decontaminate with
2N NaOH if neccessary.
Hope that helps
Regards,
ray
----------
From: Kevin Jin
I tried to use 250mM Tris pH 7.6, 1M NaCl and 5~10% 2-propanol for
dialysis. It works,
From: Taylor, Jonathan D
Dear Jerry,
I've just become aware of this spin column to remove endotoxin, marketed by Generon, UK.
http://generon.co.uk/index.php?route=product/category&path=238
Good luck,
Jon
________________________________________
----------
From: Pius Padayatti
This is in response to a comment to this thread
Kevin,
Could you explain how that worked?
How do you know your method worked?
Did you estimate the lipopolysaccharide before and after the method?
The method already mentioned here to wash using TritonX100 makes sense.
by washing bound protein sample May able to phase separate into
detergent micellar phase and get rid of endotoxins.
Most widely accepted method to separate is by two phase micellar system.
above the CMC endotoxins will be accomodated by micelles through non-polar
interactions of alkyl chains of lipidA.
Padayatti PS
----------
From: Kevin Jin
To Pius,
In my case, the protein/biopolymer is Lysine/amine riched.
1. Lys or Arg are mainly positively charged under pH 7.0 ~ 8.0, and
then provide positively charge to LPA. Of course, some metal cations
could also be involved in LPA binding.
2. Tris with the NH2 group could also be protonated and positively
charged under the same pH condition. This is why I use high
concentration Tris here.
3. Tris buffer could function as an ionic-exchange competitor for LPA
binding. If you look those commercial available Endotoxin assay kits,
they also use Tris buffer.
4. IPA could change the charge distribution on the surface of protein.
In usual, high% IPA could remove LPA efficiently but may cause protein
denature. In lot of cases, 5~10% IPA could help protein
crystallization to a larger size. As a tradeoff, it may also bring
down the packing quality.
In this case, I used it for endotoxin removal.
5. NaCl (you can try other salt) would provide additional ion-strength
to Interfere (or weak) the ionic-paring between Lys/Arg and LPA.
6. If you can add EDTA to remove metal cation, it may be even better.
In my case, I could not use EDTA.
I tested the sample (1600EU/ml). After 3 days dialysis, it went down
to < 5EU/ml. The assay kit for LPA measurement was from Charles River
(?). The procedure is too long to present here. The good thing is that
your sample would not be dehydrated and refold again.
I am saying this will work for protein in all of case, but you try in
an eppendorf tube like amini-prep.
I guess this method may have been patented.
----------
From: Kevin Jin
In my case, this method was developed for large quantity sample
(~100g/batch) purification.
Under cGMP, I could not introduce EDTA or other chemicals like
Triton-X100 into the system. Otherwise, I just solve small problem but
bring into an even large problem to the manufacture line.
I hope you can test my strategy and improve it. If you can give me a
feed back, there will be great!
Kevin
----------
From: Pius Padayatti
Kevin,
thanks.Sine this is of not much interest to many here
please feel free to go off line in the future.
The method of dialysis is confusing here.
How can one achieve this by buffer exchange?
I wonder if an extensive wash of protein on a column would work
instead.
Pius
From: Kevin Jin
Sure, we can talk about this off line.
-- Kevin
No comments:
Post a Comment