How i get clear single band in SDS-PAGE? Please help me for your clear discussion. I am looking forward for your kind help, suggestion and cooperation in this regard
The best way to get a single band is to choose conditions that obscure the undesired bands. This may or my not be academically dishonest, depending on the intended purpose of the figure/gel. You can choose to use a polyacrylamide concentration that will resolve your target band well, but will not resolve the contaminating bands well (works best for smaller contaminants). You can also load the bare minimum needed to convincingly detect the band of interest to hide the presence of the low abundance contaminating bands. However, it's generally better to get rid of unwanted bands by improving purification or expression conditions -- fix the source of the unwanted bands rather than hide them.
Hi... its difficult to get exactly single band of your desired recombinant protein on SDS page, but most of the times you can get your protein band with highest intensity(95% purity) than other proteins with Ni-NTA column chromatography... can you elaborate more your problem, what protocol you have used so far and what was the purity?
Hi,
Having single band in SDS-PAGE is a bit difficult, and it depends on at what stage you are! Do you work with cell extract or purified protein? First of all, if you are working with recombinant protein and want to overexpress it you have to use some inducer such as IPTG which finally ends to sharp band in SDS-PAGE compare to other bands. Secondly, if you are working with purified recombinant protein you must take into account what kind of chromatography you used for your protein. In some cases such as His-tag recombinant proteins it is possible to see several bands as many proteins may contain consecutive His residues and therefore they will be in different fractions. If so, you d better to use both gel filtration and affinity chromatography to obtain more pure protein and one single band in SDS-PAGE.
In any condition, if you more clarify your problem, we can more discuss it.
The best way to get a single band is to choose conditions that obscure the undesired bands. This may or my not be academically dishonest, depending on the intended purpose of the figure/gel. You can choose to use a polyacrylamide concentration that will resolve your target band well, but will not resolve the contaminating bands well (works best for smaller contaminants). You can also load the bare minimum needed to convincingly detect the band of interest to hide the presence of the low abundance contaminating bands. However, it's generally better to get rid of unwanted bands by improving purification or expression conditions -- fix the source of the unwanted bands rather than hide them.
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