In my experiment DTT removal with high efficiency and in short time is required therefore doing dialysis may not be suitable for my work. does any person know a to remove DTT with high efficiency and less than hour?
Best Regards.
Ali
There can be several approaches.Also this depends on the volume of protein solution you have
One method is that you could perform a buffer exchange using AmiconTM concentrators having a molecular weight cut off less than that of your protein. Just keep concentrating your protein by removing the existing buffer and adding fresh buffer without DTT. Perform the buffer exchange several times and your DTT amounts should go down admirably.
Another way is that perhaps you could go for size exclusion chromatography of your concentrated protein in which all your equilibration and elution buffers are free from DTT. This is just a suggestion you could look into and let me know if it works.
Another idea is that perhaps you could do away with DTT in the first place and instead work with TCEP? TCEP removal is not required prior to most applications like histidine-tagged protein purification. Of course this suggestion depends upon your downstream work. Just putting it out there.
Let me know how this works for you. Personally I feel the quickest method would be using AmiconTM concentrators if your protein volume is around 12-15ml. If more then it will take more time as several batches would be needed.
There can be several approaches.Also this depends on the volume of protein solution you have
One method is that you could perform a buffer exchange using AmiconTM concentrators having a molecular weight cut off less than that of your protein. Just keep concentrating your protein by removing the existing buffer and adding fresh buffer without DTT. Perform the buffer exchange several times and your DTT amounts should go down admirably.
Another way is that perhaps you could go for size exclusion chromatography of your concentrated protein in which all your equilibration and elution buffers are free from DTT. This is just a suggestion you could look into and let me know if it works.
Another idea is that perhaps you could do away with DTT in the first place and instead work with TCEP? TCEP removal is not required prior to most applications like histidine-tagged protein purification. Of course this suggestion depends upon your downstream work. Just putting it out there.
Let me know how this works for you. Personally I feel the quickest method would be using AmiconTM concentrators if your protein volume is around 12-15ml. If more then it will take more time as several batches would be needed.
Hi there,
I think desalting step on PD10 Sephadex G25 matrix is easy and fast (column or spin device). For more info see the following:
https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314723116657/litdoc52130800BB_20110830191706.pdf
Hi,
another possibility would be spin filter columns:
http://www.merckmillipore.com/DE/de/life-science-research/protein-sample-preparation/protein-concentration/yjCb.qB.uyMAAAE_Mgp3.Mmx,nav
Or this one:
http://www.merckmillipore.com/DE/de/life-science-research/protein-sample-preparation/protein-purification/amicon-pro-purification-system/jLyb.qB.NyMAAAFBgollvyys,nav
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