I am attempting Ni-NTA-pull down assay for finding unknown interacting partner using His tagged protein. As I'm hoping this binding partner is a small protein probably having 40-70 residues. I wanted to carry native page to detect binding of any binding partner. As SDS-PAGE show poor resolution for detecting small proteins or polypeptides. But in last of step of elution of proteins, protein interaction will be lost by high imidazole concentration. So I'm afraid that I may lose these small binding polypeptides during dialysis.
Any suggestions?
You may try EDTA or low pH elution. 10mM EDTA should not alter your proteins' ineraction if they do not bind bivalent ions. pH 6.0-6.5 may also elute you complexes, if they are not pH sesitive of course.
Have in mind that many proteins without tags will interact with your NiNTA resin and lipids/lipoprotein structures also. If you are going to analize binding partners from crude lysates you will have to wash away inpurities with imidazole. Be sure to have a control, without your protein bound to resin to see impurities bound to resin itself.
But you cant identify proteins by either gel (I mean the exact protein!) unless you have a known partner and its specific antibody. If resolving small sized protein is the only problem then I would increase the percentage of the gel (you may try 20% SDS gel or Tricine gels to resolve proteins sized 5kDa).
As said by Dawid, you have to compare always your control that doesnt have your His tagged protein in it. If you just want to know the partner and not should have to have the stable complex, it s worth a try eluting them with Imidazole.
@Dawid Deneka & Ananda Vasudevan ....I'm keeping proper controls to eliminate impurities or non specific binders...Before all this i wanted to check is their some protein binding to my protein by doing native page....then slowly i can switch to stringent washes to eliminate the non specific binders.......Thanks for your valuable input....
Hi Basvraj, my question is how will you make sure there is binding (40-70 residues!) on Native PAGE? or you are expecting bigger proteins to interact and wanna see the shift?
Hi Ananda....My protein has almost neutral pI....may be binding of new protein may change the overall pI of complex and may see the shift in band through native page.... For this i may need to try both acidic and basic page too....Yes i know it will be very difficult...But i need to give some try atleast....I'm also trying to find other mean to identify preliminary detection of binding....It would be better if i find way to resolve this small polypeptides by SDS-PAGE...Once i tried 20-25 % SDS-PAGE but resolution was bad....SO trying to find way to solve my problem....
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