protein degardation during gel filteration - (Jun/25/2009 )

I have 33.5 kDa his tagged protein purified in good amounts with little degardation and non specific bands. I dialyse the protein in 50 mM tris (ph=8). My main aim is to crystallize the protein so to remove the non specificity i tried to attempt gel filteration using G-100 column (Amersham pharmecia) with Tris buffer, the protein eluents collected after gel filteration that were giving sharp peak and high OD (1.2 at 280 nm) were not visible on running SDS PAGE. the protein might have degraded to peptidesin the column since OD of gel filterate eluents at 220 nm was much higher than taken at 280 nm. could anyone help me in this regard so as to how to prevent the degradation of protein

first, absorbance at 220 should be stronger than at 280. at 280 you are primarily reading ring structures (eg phenylalanine, tyrosine). at lower wavelengths (eg 206,214,220) you are reading peptide bonds.

is the g-100 column fresh packed? with fresh g-100?

we had a situation where we reused a gel filtration column and our protein was degraded. the column had become contaminated with a protease from a previous sample.

as a side note, you may need to increase the ionic strength of your buffer to prevent nonspecific binding of your protein to the sepharose.

2 suggestions:

1. Use 500 mM NaCl and 5-10 mM imidazole in the wash buffer along with tris/phosphate and glycerol and wash the protein bound to Ni-NTA beads with this wash buffer. THis will take care of non specific proteins.

2. Use ice cold buffers. This will take care of degradation. Alternatively, use protease inhibitors.

Hope this helps.

Best,
TC