I have purified a protein of 21 kDa by Ni-NTA. The purified protein shows a single band on SDS-PAGE but three bands on Native-PAGE. The protein is a monomer, as confirmed by FPLC. The protein also seems to be aggregating as it does not even cross the half-way mark after the dye has run out on native gel. I need the protein for NMR and immunological in vivo studies and am worried of the two extra bands in further experiments.
My protein is not degraded by trypsin hence cannot do MALDI.
Please advise.
Try checking PI of your protein and adjusting pH of the protein so it remains soluble. Also, try adding around 0.1% or less of CHAPS (a non denaturing detergent), 1-2mm DTT or 5-10% glycerol.
According to my personal experience, sometime the His tag also has an impact. The truncation of His tag sometime can help avoid oligomerization.
Also the buffer matters, like the PH value, salt concentration and reducing agents lick DTT and TCEP will definitely affect protein state and stability.
Good luck.
For MALDI you can use proteases other than trypsin (Arg-C and Asp-N) also for identifying the bands seen in the native gel.
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