5 replies to this topic

[origami]

Hi,

 

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!

 

 

 

 

[Adrian K]

Are you using syringe or chromatography system to clean your column?

How many column volume of buffer you use to regenerate your column?

Can you show us your chromatography profile?

[origami]

i am using FPLC and i do 3-4 CV regenaration.

 

 

finally, i solved my problem with the on column tag cleavage. i guess the protein interacts very high with the material of the column and finally after the tag cleavage i get the protein.

 

i tried also using peristaltic pump and i had the same results.

 

thank you very much

[Adrian K]

Glad to hear that. Congrats.

Just curious, based on my understanding that we need to do at least 5 CV for washing and regeneration. Any reasons to share on using lesser CV as you did?

 

[origami]

yea, i thought the same and i used to do before like this but i didnt see any difference between 3-4 CV to 5-6 CV.

[littlebull]

Your protein either does not binds to the column (e.g losing tag) at all, or weakly binds to the column.

 

1. no binding: most of your protein is in flow through, the rest aggegates on column which was washed away by 0.5 NaCl.

 

2. weak binding: you can load your protein twice, or add resin to your protein solution and incubate for a few hours or even overnight.

 

Good luck