Protein purification wash problem - (Apr/26/2006 )

I am purifying my protein that is expressed in E. coli under denaturing conditions. My wash buffer is made of:
50 mM sodium phosphate pH 8.0
10 mM Tris-HCl pH 8.0
450 mM sodium chloride
50 mM imidazole
6 M Urea

I am loosing a large amount of protein during the first wash step. Any suggestions to eliminate this problem?

You could try to reduce the concentration of imidazole. Imidazole is competing with your protein to bind to the column and it may be successful at doing so at 50mM. Every protein is different and may not need up to 500mM for elution. You could try stepping it up from 5mM to 50mM during your washes and elution. If you are worried about the binding of non-specific proteins then equlibrate the column with buffer + imidazole prior to binding your protein.

Remember pH also plays a significant role in eluting proteins. You must make sure your wash buffers' pH is not too low as this will protonate your his tag and subsequently make it impossible for your protein to stay bound to the resin. Common pH's used to elute proteins are between 4.5 and 6.

Hope this helps.