acetylmandarin

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From my research, I found that SDS gels do not break covalent interactions. So how can you say that C is valid in this case? I see what they are trying to say---since the variant in Gel A (the SDS gel) has a band at a lower MW than the variant well in Gel B (native gel), this means that the variant contains multiple subunits which were broken apart by the SDS. However, I thought non-reducing SDS was only able to break non-covalent bonds.
 

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acetylmandarin

acetylmandarin

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Oct 20, 2014
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Actually, in C, are they comparing the wild type to the variant? In the SDS gel, the variant runs as 70 Da, while the WT shows two bands, one at 50 and one at 20. Since both are made of 140 Da total, the WT must have two bands at each band shown (so 4 total), and the variant band must be two bands total, overlapping, since each is 70 Da. They are made of covalent interactions in the variant since they are not broken up by the SDS gel.
In the native gel, the subunits of the variant are somehow associated via non-covalent interactions, since the variant on the native gel shows a single band at 140 Da.
 

aldol16

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SDS is a detergent. It coats the protein with a uniform negative charge. So it breaks non-covalent interactions like dipole-dipole interactions and ionic interactions. In this case, you're asked to compare the WT to the variant. C say the variant has covalently-linked subunits and this is correct because under SDS-PAGE conditions, the non-covalent interactions are broken in the WT and the subunits are further broken apart into the 50 and 20 kDa units. In the variant form, these subunits stay attached to each other with SDS treatment, meaning that the interactions holding those subunits together in the variant are covalent.
 
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acetylmandarin

acetylmandarin

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Oct 20, 2014
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SDS is a detergent. It coats the protein with a uniform negative charge. So it breaks non-covalent interactions like dipole-dipole interactions and ionic interactions. In this case, you're asked to compare the WT to the variant. C say the variant has covalently-linked subunits and this is correct because under SDS-PAGE conditions, the non-covalent interactions are broken in the WT and the subunits are further broken apart into the 50 and 20 kDa units. In the variant form, these subunits stay attached to each other with SDS treatment, meaning that the interactions holding those subunits together in the variant are covalent.
Thanks. I think was initially confused me about this was that I forgot that both the WT and variant were of the same 140 kD protein, so I didn't realize they were directly comparable like that.