His-Taq purification - (Sep/14/2011 )

i have expressed HtrA protein into pTaT vector harbouring E.coli BL21.The protein is in periplasm then havested and breaked cell by sonication, centrifuge and step to purify protein with Ni-NTA (Quigen). My problem is in step of purification . my protein is very low yield after wash ni bead with washing buffer (20 mMTris, 250mM NaCL, Triton X 100 5% w/v , 20 mM imodazole). Thank you for your recommend and Forgive me in english writting.

Hola, periplasmic expression vectors are designed to have a clean periplasmic protein fraction at the first purification step. If you sonicate you will have two protein species: the processed periplasmic one and the non processed cytoplasmic , wich has a Nt signal peptide. If your construction has the histidine tag after the Nt SP and you have your expression induced could occurs that you see a lot of protein that not bound the resin. if your tag is Ct i haven´t explanation. But think a thing more, secretion systems in all organisms have a handycap you are increasing your recombinant protein expression, that has to be processed by the normal processing machinery wich is easyly saturated, so it ´s better a light induction in a long period before harvesting.
Look for the osmotic shock method for your purification and one more aspect, I think that the amount of detergent in your buffer is a bit high. Buena suerte

The His-Taq is at the N-teminal of my recombinant protein . Now i used pTaT vector because I used to expressed in pET28 vector and my protein is form to inclusion body. and in pTat vector i have successful to production protein in soluble form. My problem is purification step. My protein is still binding with Ni-nta bead when wash by buffer 1 Tris, NaCl Imidozol 10 mM and triton x 100 cause i have run SDS-PAGE my protein still intense and single band. but then i wash with buffer 2. my protein is a little. Why I solve this problem? and thank you for your recommend.

Addition, I have purify protein bring to produce antibody.

Hello, I have a question. I have quite similar problem. I want to purificate my protein with the beads (with bound antibody). I dont have the comercional washing buffer, so I use the blocking buffer. But my beads are always destroyed. I would like to ask, if you know, what can degrade the beads and how long (and in whitch) can be the beads with the bound protein stored (I use the comercional storage buffer with Na3N)? Thank you for the answer.