Hi

you can try to elute by using

100, 0 mM NaH2PO4

10, 0 mM Tris-HCl

8, 0 M Urea Adjust pH 4.5

you can increase the elution time overnight and at 4°C

best regards

can you specify your m.o.?

the beads where you see the protein in an SDS-Page gel (i presume) are the sepharose beads of the chromatographic matrix?

how do you promote the contact between the matrix and the protein suspension? (it can be a binding problem) you can do a batch contact overnight and then pack the column and proceed with the IMAC

do you check the GndHCl buffer pH?

You elute the protein before or after refolding?

@Carlos Lopes

I am doing Ni NTA purification. I am doing PAGE followed by western blotting. I can see protein on beads but not in elutions. Protein is binding to Ni Beads actually.

@Bogdan Munteanu

40mM Tris, 0.01% NP40, 10% glycerol, 150mM NaCl, I used this for elutions

do you think EDTA can help ?

Thanks

Hi Paras,

never tried,

you can try, and perhabs it might help

please find a really good handbook for protein expression and purfication from HisTagged proteins

https://www.qiagen.com/de/resources/resourcedetail?id=79ca2f7d-42fe-4d62-8676-4cfa948c9435

if you add edta you will wash out the nickel from the matrix... and whatever is binded to it. you can try just to check if your protein in still there or if it is washed by any reason in you washing procedures...

if you refold the protein in column it can be washed out during the refolding due to the inaccessability of the his-tag...

https://books.google.com/books?hl=pt-PT&lr=&id=zTiRJHpKIQoC&pgis=1

https://books.google.com/books?hl=pt-PT&lr=&id=zTiRJHpKIQoC&pgis=1

This question seems to be asked many times on RG.  In most instances this involves the protein precipitating/aggregating on the column in cases of cysteine containing molecules that require proper folding.  Binding in this case is due to insolubility rather than His tag even in the presence of chaotrope.  You unlikely have reducing agent present due to Ni reduction and therefore a cysteine-containing protein can oxidizes to an insoluble mis-fold through improper intra and inter disulfide bridges.  If EDTA, acidic pH, Ni reduction, etc. don't cause the release of protein then it may be just highly aggregated.  If you had to remove protein bound to the IMAC resin by treating a sample of the protein-bound resin with Laemmli buffer in order to run the gel analysis for bound protein then that process may answer your question.