i am working with a N-strep tagged, cleavable by 3C, hydrophilic protein and i see it on the FT and after regenerating the column (GE healthcare, 5 ml) with 0.5M NaOH i see again the protein.
i thought that:
-either the affinity (material-protein) is too strong,
-or my protein is aggregated (i did SLS with the eluted protein after the regenaration and the peak and MW corresponds to the protein, therefore i excluded this thought),
-or the concentration of the loaded protein is too high (the lysis buffer was 1/100 and i filtered before loading it onto the column, therefore i excluded this thought),
-or the flow rate is not the proper (i tried with 3ml/min and 5ml/min, the result was the same, therefore i excluded this thought)
-or the strep tag is hidden (i add to the lysis buffer up to 10mM b-merca, therefore i excluded this thought)
-or the d-besthiotin was not enough (i add up to 10mM d-besthiotin, therefore i excluded this thought)
-or the pH of the buffer was below 7 (i checked the buffer and it was fine)
-or to increase the NaCl (up to 250mM, again the same result).
my last hope is to do the tag cleavage on the column overnight and hopefully the next day to get my protein in the flow through. Additionally, to add up to 1% Triton X100 to the lysis buffer.
any suggestion is wellcome!