I am investigating the ProteinSimple Simple Western system, but have found that my proteins (and some ladders that I've used in the past) come out at very different molecular weights compared to (traditional) western blot. If you've used the Simple Western, did you have the same experience? How did you address this in your publications?
I have experience working with ProteinSimple Wes, but not in publishing results from Protein Simple Wes. I have observed that the apparent molecular weight of some proteins differ from the expected/calculated monoisotopic mass and from the mass that is observed by PAGE.
However, it is not uncommon to observe between the expected and observed mass of a protein by any of several separation methods (including PAGE, or PAGE with different pore sizes/concentrations).
I believe that this separation artifact can be dismissed using evidence from direct and indirect mass determination (e.g. via expression vector DNA sequencing, protein mass-spec analysis or dynamic light scattering).
ProteinSimple's customer service is generally excellent. I imagine they can offer advice/insight on how to mitigate these concerns.
Thanks for your response Brian. Were the differences that you saw very large? I’m seeing a difference of 85 KD via SDS page to 110 KD on Wes and that’s just such a big difference from what we’ve been running, and what the literature give. None of my colleagues will accept that much of a difference.
Did you ever ever try to run another company’s Ladder on Wes?
The differences I've observed have been much smaller than that, so I can appreciate your concern. I've never tried to run multiple biotinylated ladders via Wes.
I still think that separation independent methods are a more reasonable means of determining the protein's mass. Perhaps the protein being analyzed has unusual properties than are affecting the migration.
Are your flourescent standards separating similarly in both the ladder and the experimental lanes? I've often seen that the low molecular weight fluorescent standard in the ladder resolves as two bands, which the software 'calls' as two separate standards while ignoring the actual middle flourescent standard. This discrepancy can cause a rather large shift in the apparent molecular weight of the experimental lanes if the low molecular weight standard resolves as one band in those lanes. You can manually correct the assignments by right clicking in the standard lane view and either telling the program to ignore a standard, or to manually assign a standard.
- Badges
- Science method