Hi Joseline,

I use for washing buffer 0.1M TrisHCl, pH8.0 with 0.15M NaCl and for elution the same buffer with 10mM desthiobiotin and it works perfectly. Maybe try to increase the concentration of desthiobiotin.

I found in the IBA material : When using Strep-Tactin® Superflow® High Capacity resin it may be advantageous to use 5-10 mM desthiobiotin to get the target protein eluted at higher concentration (especially in combination with Twin-Strep-tag®).

Hi Milan,

thanks for your quick answer. The manual for the spin columns states that one should use D-biotin i.o. desthiobiotin to get higher concentrations when working with the spin columns (as they're single use). I've tried both and neither works at 2 mM. However with my FPLC Streptactin column I am able to purify protein with 2 mM desthiobiotin. As in theory it should be the same material, I don't understand why it doesn't work for spin columns. However I will certainly try your suggestion!

Did you let it stand for a while or have you spun it immediately after addition of the elution buffer? The incubation could help.

The binding of biotin should be basically irreversible, that's why one should use desthiobiotin instead, so it's weird your protein binds even stronger. Are you sure the binding is specific? Didn't your protein just precipitate?

Hi Tomas,

As you said, the biotin should be irreversible, that's also why I find it so strange that my protein won't elute from the spin column.

I've tried both spinning down immediately and letting it stand for a while (>10 min). Didn't make a difference in the amount that I eluted.

Precipitation might be an option, though as I know the protein itself is very stable and I've been able to purify it before with Streptactin, I would find it unlikely... 

So I've tried higher biotin concentrations (10 mM), this doesn't lead to higher elution.

I've also checked for precipitation by washing the top of the column after the elution steps. There doesn't seem to be any protein on top of the column, so I don't think it has precipitated there.

I also got a tip from IBA, the producer of the spin columns to lower the pH to 6 ~ 6.5. There is a Histidine in the Strep-tag and lowering the pH could break the bonding with the streptavidin. Unfortunately, lowering the pH does not help either.

I have attached a file with a blot probed with StrepMAB Classic.

If anybody has any other smart ideas, please let me know!

Hi, I am also having the same issue. My protein also binds to the resin and I am not able to elute it even with 10 mM desthiobiotin.

Were you able to solve the issue??

Any suggestion would be appreciated.

Thanks a lot.

Hi, I am also having the same issue. My protein also binds to the resin and I am not able to elute it even with 10 mM desthiobiotin.

what's the role of the EDTA, I didn't add it in the elution buffer.

Were you able to solve the issue???

Any suggestion would be appreciated.

Thanks a lot.

Why does my Strep-tagged not elute from Streptactin spin column? - ResearchGate. Available from: https://www.researchgate.net/post/Why_does_my_Strep-tagged_not_elute_from_Streptactin_spin_column [accessed Mar 11, 2017].

Hi,

sorry for the late reply! In the end I gave up, I wasn't able to elute the protein with any of the advised conditions from the spin columns. However when I used the FPLC column resin, the protein behaved perfectly (elution with 2.5 mM destihiobiotin). So I think it had something to do with the material they used for the spin columns.

Cheers!

I recommend pH-ing your 5-10mM dethiobiotin before using it for protein elution. If you simply dissolve the powder in your elution buffer, you are likely way below your assumed pH, which will interfere with the elution process. Good luck!