Hello Everyone,
I am looking to purify Recombinant tau monomers for my studies. But we don't have FPLC system with us. I am wondering any methods available to purify without the FPLC system. Please kindly share your answers.
Thanks
Mani.
I would suggest bacterially expressing the recombinant protein with a GST or other tag so you can purify by affinity chromatography and cleave off the tag
Kristin M. Adams Thanks for your suggestion and if you have any protocol or papers, could you please share with me?
Samidurai Manikandan I'm having issues getting access to the full article but here is a start:
https://pubmed.ncbi.nlm.nih.gov/18429193/
First step would be to clone the DNA coding for your protein into the pGEX-2T vector. You will express this under an IPTG inducible promoter in BL21 bacteria, lyse the bacteria, and run the lysate containing your recombinant and GST tagged protein over a glutathione sepharose 4B column, and elute the protein off the column using thrombin which will cleave the protein from the GST tag and leave you with only your recombinant protein.
spy tag spy catch method maybe we can try Article Spy&Go purification of SpyTag-proteins using pseudo-SpyCatch...
Dear Samidurai Manikandan
i think that you can clone it with -N- or C- terminal His tag (you can also add a TEV protease digestion site to remove it after purification) and perfrom the IMAC purification using hand made gravity flow coloumns assembled by insert bulk resin in a empty coloumn.
Since there is His-tagged commercial avaialbe Tau protein
https://www.sinobiological.com/recombinant-proteins/human-tau-10058-h07e
i suppose that the tag is not a problem for the Tau protein,
Depending from the amount of protein that you would like to purify you can use different kind of coloumns,
eg:
BIorad Poliprep coloumn (cod 731-155) with 200-300ul of Ni-sepharose FF resin (Cytiva)
1-5 mg --> PD-10 empty coloumns with 1ml of Ni-Sepharose FF resin.
in generally i'm using
Tris 20mM, imidazole 10mM, NaCl 300mM pH=8 as lysis/bindng buffer
Tris 20mM, imidazole 20mM, NaCl 300mM oH?8 as wash buffer
Tris 20mM, imidazole 300mM, NaCl 300mM pH?8 as elution buffer
you can follow the different step by checkng the drops that coming out from the coloumnn with the bradofrd reagent (mix 10ul of sample with 100ul of bradfrod 1X)
you can find some more infromation at
https://proteocool.blogspot.com/2021/05/tips-of-week7small-scale-imac.html
in this case i report the methods that i'm using for purification from mammalian cels surnatant, but it is the same from soluble fraction extracted from E.coli after lysis by sonication.
best regards
Manuele
Tank you Manuele Martinelli Kristin M. Adams Subramani Karthikeyan for your answers.
You can try to add a purification tag when recombining expression, and use the corresponding affinity column for affinity chromatography. The purification efficiency of the whole process is high. If you accept customized recombinant proteins from merchants, maybe CUSABIO is a good choice. CUSABIO provides protein expression services starting from whole gene synthesis. There are five major expression systems to meet the needs of different experimental proteins. You can find more details on this page: https://www.cusabio.com/protein_service/
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