I am trying to do a thermal shift assay for a recombinantly-produced membrane protein, by western blot band quantification. I use Bio-rad 10X Tris/glycine SDS running buffer for my SDS-PAGE and dilute it down to 1X. Out of a suggestion to me, I did an experiment where, instead of diluting it down to 1X, I used 2X buffer instead. Could I please ask how increasing the running buffer concentration affects proteins that you're running? The 2X running buffer made known contaminant peptides show up brighter on my western, as well as my protein of interest, and my protein bands were more intense in general. There seemed to be an increase in signal. Does anyone know the chemical explanation for this? Maybe more denaturation of proteins and thus more exposure of their epitope that the primary antibody can recognize? I don't know.
Also, I have seen a smearing of sorts at the top of the western membrane in EVERY lane. It has continuously puzzled me, because I cannot figure out what it is. I skip wells when loading samples in order to space them out, and I see the smears even in the empty lanes!! My primary antibody is kind of dirty and nonspecific (it even binds to my protein ladder). I thought that stuff at the top might be nucleic acid contamination from the protein production, but it looks like it coincides with the top 3 molecular weight markers on my protein ladder, but in every single lane. It's not like I'm not changing tips after loading my ladder first, and then loading my samples. I change tips every time. Also, I don't see how the ladder could migrate sideways through the gel, so the "smear" being part being from the ladder sounds like a preposterous idea. When I used 2X running buffer, the "smears" got sharper and I was able to make out whatever that mysterious stuff is, and again, it looks like the top 3 molecular weight markers on my protein ladder (i.e. 250, 150, 100). Or I guess it just looks like pretty evenly-spaced bands at the very top. I would think it's sample contamination of some kind if it wasn't in the empty lanes. I tried looking at the Rice University SDS-PAGE Hall of Shame website, but I couldn't find an explanation.
These observations I'm having are very weird, and I was hoping someone might be able to shine some light on them. Thanks!
Have you attempted running just your ladder and a negative control to check if you'll get the same consistency? Your running buffer might be contaminated
First of all, check the SDS-PAGE buffer for contamination or precipitation. You can try making fresh 1X buffer using Tris, Glycine, and SDS and use it for running the gel. Using 2X buffer will not only increase the SDS concentration in the buffer but also will increase the ion concentrations, which might be the result of what you are observing.
The smearing that you are observing might be the result of either excessive boiling of the sample or a high concentration of your sample buffer. Or if you are observing the smear while developing the blot that might be the result of non-specific binding of your primary antibody. So to rule out that possibility you need to load a negative control in one lane of your gel.
Hope these suggestion help you.
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