Because of I would like to vary condition for NiNTA purification from pH 2-9 but when I dissolve cell with low pH buffer and then sonicate it. Flocculation was happen. After I centrifuged cell lysis and then measure protein concentration it showed very low and then run check protein in SDS show nothing because almost was flocculate.
Most cytoplasmic proteins are insoluble at acidic pH because they are denatured. The denatured proteins aggregate and precipitate, as you observed. You should not use such low pH. Stick to near-neutral pH, at which cytoplasmic proteins are soluble.
If you are interested in lysosomal proteins, a lower pH is reasonable. Then, you should begin by purifying lysosomes.
As far as I know, lower pH (>5) elutes the bound protein. I think it is not advisable to go for a pH lower than 5. Imidazole usages is preferred over lower pH to elute bound proteins. As earlier method allows protein purification in native form (in most cases). Hence under such conditions, his tag may not be able to bind NiNTA.
Thank you for your suggestion.
yes I know that if low pH it not allow his tag bind with Ni-NTA but when I did it it remain a small amout of bind protein from this I would like to check it. if lower than pH5 it will more decrease or not
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