Sure, we can talk about this off line. -- Kevin
On Thu, Mar 8, 2012 at 7:48 PM, Pius Padayatti <ppadaya...@gmail.com> wrote: > 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 > > On Thu, Mar 8, 2012 at 4:01 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 > > > > -- > Pius S Padayatti,PhD, > Phone: 216-658-4528
