I am currently looking at dose dependent effects on protein expression using western blot analysis. So far, I have been getting inconsistent trends in expression and a significant degree of variability, though care was taken to eliminate the variabilties in advance
Western blot signal variability is typically due to either inconsistent protein sample loading or non-linearity of the detection system. Using a control that you anticipate will not vary (e.g., tubulin) can help to normalize extract loading between samples. To determine the linear range of your detection system you can load a range of protein concentrations, quantify the resulting western signals and then determine the range of protein in which your assay is linear. You can then adjust the amount of extract you load to ensure your anticipated changes are detectable. Without more details, It's hard to give more specific advice.
Western blot signal variability is typically due to either inconsistent protein sample loading or non-linearity of the detection system. Using a control that you anticipate will not vary (e.g., tubulin) can help to normalize extract loading between samples. To determine the linear range of your detection system you can load a range of protein concentrations, quantify the resulting western signals and then determine the range of protein in which your assay is linear. You can then adjust the amount of extract you load to ensure your anticipated changes are detectable. Without more details, It's hard to give more specific advice.
Thank you Ken for your advise. I actually used beta-actin and GAPDH as loading control. Surprisingly, they didn't show similar trends, and sometimes, opposite trends. I excluded tubulin as a loading control because I suspect my treatment to affect it in some way. I ensured equal loading by running BCA protein assay on my samples prior to adding the loading dye and running the gels. Before quantification, trends of the dose response do not reproduce as predicted.
If you are confident your western blot is linear in the range you are measuring, then perhaps your variability lies in the treatment you are measuring. Do duplicates of the same treatment also show variation on the western blot? This would point to an experimental variable that is not being controlled. Good luck!
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