Dear All, This may be slightly off-the-track question but your feedback will be very much appreciated. The situation is-
I obtain a very low amount of the protein of my interest (a hetro-dimer) from the construct I am using (only 8% of the total amount of protein obtained is the protein of my interest). After 2 column purifications (Ni-NTA and St) the concentration of the protein is around 0.24 mg/ml (volume- ~1.0 ml) from a litre of bacterial culture and in ~300 mM NaCl present in the elution buffer. To reduce the high amount of salt I have I use a desalting column which, further lowers the protein concentration significantly. I need atleast 1.0mg/ml of protein concentration and to the amount of ~200 microlts for further experiments. As the last resort I try to use high amount of bacterial culture (~6lts) to scale up the yield and use centricon to concentrate the protein at various stages. I am partially successful to obtain 0.56mg/ml of protein concentration and up to 50 microlts of it. Another problem is that the protein is notoriously prone to aggregation (> 1.5 mg/ml), so dialysis for ~ 2 hrs or so to reduce the high salt concentration has failed miserably. Please do send your feedback. Thanks and Best Wishes Andy Dr. Anindito Sen (Ph.D) Department of Cell Biology & Anatomy Graduate School of Medicine University of Tokyo Tel & fax: +81-3-5841-3339
