Good morning,
I am purifying a thioredoxin mutant (but behaving wt-like) from coli, with its unique Proline labelled for NMR (13C and 15N).
On Friday I lysed the cells and collect the soluble fraction, in 20mM TRis HCl pH 7.5 (at RT). I then froze the lysate in liquid nitrogen and stored it at -20°C.
Yesterday I thawed the lysate for dyalisis before anion exchange column. the lysate was cloudy. I thought that maybe could be because of a shift in pH so I dyalized in Tris buffer 20mM ph 7.5 at 4°C (the final pH was so 8). This morning the same story. I try to dyalize at room temperature, so that the pH was 7.5. Nothing better. Now I'm spinning down everything and I will load a gel with the cloudy lysate, the pellet and the soluble fraction.
Does anyone know why this could be? and Does anyone have a suggestion on how to rescue my protein?
Your buffer is not suitable for the solubility and stability of the proteins in the lysate. You need to identify the optimum buffer conditions that maintain your proteins in their soluble and stable forms. What is PI value for your target protein/proteins? Try to look for identify the pH range, salt concentration, solubilizing and stabilizing additives for your lysis buffer conditions. You may need to use protease inhibitors depending the sensitivity of your proteins to protein degradation.
Thank you for your reply!
Well, what actually sounds strange for me is that this is not the first time I'm purifying this protein.... and I never had this problem! the PI is 4.7 and I have protease inhibitor, PMSF and DNAse in my lysis buffer.
Did you change your expression conditions, particularly Temperature and IPTG concentration if you have used the bacterial expression system? Did you run the total cell lysate and the soluble fraction on SDS-PAGE? Did you detect your target proteins at their expected size range? Did you use fusion tags? You may need to check your expression plasmid?
I just changed the temperature because of the labelling protocol, but the protein is usually well expressed (I decreased the temperature from 25 to 18 degrees this time). My protein is not present in the soluble fraction of the lysate, and the pellet is only protein aggregates ( :( )
I don't have any fusion tag and I used the same cells as the last purification with the same plasmid.
It looks strange this time if you are able to purify it previously. Double check the contents of your lysis buffer. Try to prepare the fresh one. It is also good to check your plasmid for possible mutation of your target gene. some times, changing a single amino acid may be sufficient enough to change the physicochemical properties of a protein.
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