We just bought the new strep-tactin XT for protein purification. The manufacture recommends to use 50 mM biotin to elute the protein. While 2.5 mM desthiobiotin is suggested for protein purification when using strep-tactin beads. I used 2.5 mM desthiobiotin to elute protein from strep-tactin XT but no target protein was purified. Does anyone have similar problems?
Thanks in advance,
May be your tag is not be accessible. Yes 2.5 mM is what they suggest, try to elute more like 10 CV. If you have other tag you may try that one, or buy bulk resin and incubate longer
Hi Mahesh Chandak , thanks for your suggestion. The new strep-tactin XT is suggested to use 50 mM biotin (not sure why they suggest such high concentration of biotin) to elute the protein but I used 2.5 mM desthiobiotin. Just not sure if it is sufficient or not to elute the desired protein.
Usually 2.5 mM is what they recommend, but you may try 50 because manufacturer is suggesting 50mM, Later you can do size exclusion and check your protein
Thanks Mahesh Chandak After running a SDS PAGE gel, it seems that most of the proteins are not binding to the column. lots of them present in the NaOH strip solution. Maybe the tag is not accessible as you mentioned before.
Desthiobiotin is totally incompatible with the new XT system. Do not even attempt to use it. According to the manufacturer:
"Strep-Tactin®XT requires elution of the target protein with Buffer BXT containing 50 mM biotin (Due to the high binding affinity desthiobiotin cannot be used for elution.) "
I don't understand why "high binding affinity" would discourage elution since, hypothetically, the whole point of adding the biotin/desthiobiotin is to displace the strepII tag from the resin (Therefore, one would expect that desthiobiotin would be superior to biotin for the purposes of elution). Regardless, it's usually better to just do what the manufacturer suggests since they've spent years perfecting the system.
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