large quantity of high concentration protein needed - (Mar/16/2008 )

hi all,

i have produced my recombinant protein using ek/lic vector from novagen. all is well with the protein i purify using NTA resin and it comes up as a lovely clean bright band on SDS PAGE gel. however when i bradford the protein *after concentrating* on amicon filter i get only 240ug/ml (concentrated 10mL to 1mL).
the only thing is i need large volumes of high concentration protein (5mg/ml) for FTIR and i am really struggly to achieve this. has anyone any ideas on concentrating this protein. i really dont want to be using amicon filters they very expensive and time consuming. im thinking im gonna need to scale up big time to L volumes. (had been using 500mL cultures) any ideas?

many thanks!!!!!

Hi,

Maybe you can try manual precipitation with salt (eg:ammonium sulphate etc.) and cleanup with dialysis tube.My friend used it and get a large amount of purified protein.But of course, must know everything about the protein before you can manipulate it.Good luck.

How does the size of your protein compare to BSA (which is what I presume you are using for the standard curve)? If there's a significant difference, your calculation might be skewed. Try running on an SDS_PAGE gel system, and compare with BSA.

Do you rinse off the sides of your spin filter? Occasionally, the protein can stick to the filter if the concentration gets too high. Take a few time-course measurements of the OD while you concentrate it to see if you're losing protein onto the sides of the filter unit.

try an ultrafiltration unit by centrifuge method. Pull, Amicon make these.