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 On Thu, Mar 8, 2012 at 1:01 PM, Kevin Jin <kevin...@gmail.com> wrote: > 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. > > > > > > > > > > > > > > > > > > > > > > > On Thu, Mar 8, 2012 at 7:16 AM, Pius Padayatti <ppadaya...@gmail.com> wrote: >> 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 >> >> >> >> On Wed, Mar 7, 2012 at 9:50 AM, Jerry McCully >> <for-crystallizai...@hotmail.com> wrote: >>>o >>> >>> 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 >>> >>> >> >> >> >> -- >> Pius S Padayatti,PhD, >> Phone: 216-658-4528
