You have a large amount of protein for deposition before purification. Try to reduce to see if the double band is present or not. If it is present, it could be an artefact of the SDS-PAGE assay rather than a feature of your protein/process.

I find that using SDS sample buffer and heating to 95°C (5 min) for denaturation causes di-sulphides to shuffle in my protein and therefore give me doublets in my non-reducing gel.

I switched to LDS buffer and heating to 70°C (10 min) prevented the doublet from forming but still gave sufficient denaturation of the protein.

Or check both bands using a mass spectrometer, maybe one of the bands is a cleaved N-terminal part of your protein of interest.

DEar Rithika Ashokan

what are you removing with thrombin digestion? his-Tag?

There are 2 possibilities:

1) You have oly partial digestion and therefore the upper band is undigested while the lower is digested

2) So you detect the band of the thrombin

however to provide a better answer i need to know more about the loading order of the gel and of the MW of the protein marker

best

Manuele

Manuele Martinelli I'm trying to cut his tag. Yes, sir, you are right, actually, the protein was partially cut, hence I could see the thrombin in the flow through, wash through even in 100mM NaCl elution.

Loading order, Marker, Imac 100mM imidazole fraction, Dialysis (digested), FT ion exchange, WT ion exchange, 100mM Nacl, 300mM NaCl. 500mM Nacl, 800 mM NaCl.