BioMed Research International / 2019 / Article / Fig 5

Research Article

Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures

Figure 5

Example of a Western blotting experiment that could be used to estimate differences in protein abundance. (a) Fluorescent image of the membrane showing proteins in each lysate that were labelled with Cy5. SeeBlue Plus2 Marker was used for sizing (not shown). This image was used to estimate the total protein abundance for each sample. Arrows indicate the approximate location of ENPP1 (open arrow) and Fam3a (closed arrow). (b) Western blot images of recombinant ENPP1, and Fam3a that were used to compare their abundance. These proteins were detected by chemiluminescence. (c) The standard curve of recombinant ENPP1 O.D. values that was used to interpolate levels of recombinant ENPP1 in each sample. (d) The standard curve of recombinant Fam3a O.D. values that was used to interpolate levels of recombinant Fam3a in each sample. (e) This table shows the results of interpolating the O.D. data against the standard curve for that protein as well as the comparisons made using the raw O.D. data.