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
