Depending on how big your broccoli is I would simply crush them and mount as
big of a piece as possible and see how well it diffracts.
I assume you have tried already:
- glycerol
- changing protein:reservoir ratios
- temperature
- adding oil to either / and the reservoir, your drop
-crushed up some broccoli and recreened your original sparse matrix screen
while seeding with chunks of broccoli seeds
- when you say the His-tag needs to be cleaved otherwise it will precipitate,
that mitt also just be either leaching Ni from your column or simply the
imidazole
- You don't say how you purify your protein, I hope you are not just setting up
after the Ni capture step and at least have some other method following e.g.
anion or SEC
Just a few incomplete suggestions,
Jürgen
On Aug 26, 2011, at 6:56 AM, eswar reddy wrote:
Dear All
I was working on a Human protein and expression and
solubility is good in E.coli and purification is One step (His-Tag), and i
need to cleave the Histag before screens, if not the protein will precipitated
and Aggregated, but after trying for 1.2 years i have crystals and they are
with Organic solvents, (10 conditions), these crystals are inter grown like
broccoli shaped and i tried seeding, but it is not successful, and even i
tried with additive screen but the result is the same .... is there is any
idea to increase the size and shape of my protein crystals.
Any suggestions will be helpful for me
Thanks in Advance
......................
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-2926
http://web.mac.com/bosch_lab/
