Following purification of two different recombinant proteins (at the same time, in the exact same conditions and same buffers), I have made the following observation: while the same concentration was measured by bradford assay, the signal intensity on coomassie staining of the proteins after SDS page migration showed a two fold difference between my two samples. Since the bradford assay is coomassie-based, the two assays are biased towards the same amino acids. Could anyone provide me with an explanation for the difference observed? And which assay should I trust then for further experiment ?
Thanks in advance for your help
Antoine
There are multiple reasons for observe different results in SDS-page gel and bradford assay. First, bradford mesure the quantity of total proteins in the sample and in SDS-page you look only the band corresponding to your protein. Are the two proteins 100% pure in the gel? if there are contaminants this can explain the difference. Another explanation is the molecular weight, have the two proteins aproximately the same MW? Usually, when different proteins have a big difference of molecular weight coomasie blue stained gel can show differences in the band.
Hi there,
The difference between the two techniques is the fact that in SDS PAGE you measure proteins which have actually entered the separating gel. In case of partial aggregation in your protein sample, some of it might stay stuck in the well or migrate like HMW protein. Then for the sample giving less signal than the other it might be that the protein is forming soluble aggregates.
Hi
I recommend use you silver stainning in SDS PAGE because it is more sensitivity than Goomasie.
In addition to previous answers, the differences may arise from interfering substances in your buffers. For instance SDS can artificially increase the signal in the Bradford assay and therefore mask the differences between your samples.
See here for the mist of interfering compounds
https://tools.lifetechnologies.com/content/sfs/brochures/TR0068-Protein-assay-compatibility.pdf
Hi!
I suppose the molecular weight of your proteins is different. I have observed that running the same quantity of proteins with different molecular weight; high molecular weight proteins run clearly, and the band is more thin, meanwhile, proteins with low molecular weight, are most thick, because it is most resolving. I hope this be usefull.
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