I have purified overexpressed protein from BL21 (DE3) cells using Ni-NTA column. When we run purified protein through native PAGE and SDS PAGE both which showed different result. In SDS page showed only one band of purified protein whereas two band in native PAGE. I have proceed whole experiment three times and found same results. What is the possibility to find two band in native page whereas it one in sds page. I have attached native PAGE image.
I suggest you to have a look at earlier discussions related to your question:
https://www.researchgate.net/post/Can_anyone_detail_the_differences_between_Native_PAGE_and_SDS-PAGE
In short: SDS page distinguish due to their set up primarily on size/mass differences of proteins present, while positions of protein bands in native page is influenced by the proteins’ structure and charge as well.
I cannot judge based on what you included in which range we are looking at, so it is hard to say on what differences in size we are looking at, but I suspect that the explanation is:
You’re looking at two different aggregation states of your (pure) protein (for example caused by the presence of disulphide bridges).
Hope this answer is of any help to you.
Hi there,
I don't know exactly what is your protein and the size of each band. But I guess due to dimer forming in native gel (non-reducing), then there are 2 bands but only one in reducing gel (SDS page).
Hi, Shweta,
Could you give us some more information about your protein and present the SDS-PAGE result as well? Does your protein contain Cys residues at all and if the answer is yes, is there an odd number of them (1, 3, 5 ...)? If yes, then the explanation of Phuong Hoang is likely to be correct (but keep in mind that in native PAGE you cannot simply deduce protein sizes from standards as they might behave differently in absence of SDS). In the opposite, it is still possible to see a dimer on native PAGE as monomers can stick together by other interactions than covalent. SDS would break these bonds, so you see just one form, and absence of SDS reveals two protein species.
One can't fully exclude another possible reason for seeing two bands on native PAGE: in the case your protein is relatively large and/or you eventually overloaded the gel on SDS-PAGE, you might have missed a partially degraded protein, lacking e.g. 5-10 residues. If these residues are charged, their absence could result in a substantially different net charge of your protein. The shorter version of your protein would give you an additional band on native PAGE.
What could you to to better understand the presence of two bands in native PAGE is (a) run analytical gel filtration in the range of sizes where your mono- and dimer are expected, and/or (b) run 'reducing native PAGE' (i.e. include the same concentration of 2ME as you did in SDS-PAGE) and compare with the true native PAGE. Hope this helps.
Its dimeric protein as for my knowledge about the pritein you are intrested posses cystin. Thereofre, dr.marko is correct.
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