Figure 3
Quantitative western blot workflow summary. (A) Optimization of the assay for the specific set of samples using a specific target. Step 1: Sample Preparation and Quality Control Testing. Produce a pooled lysate from all treatments to a maximum initial concentration of 4 µg/µl and produce a standard curve by diluting serially by a factor of 1:2 for seven dilutions loading 20 µl per well on an SDS-PAGE gel. After gel-based separation, transfer to membrane using a consistent transfer apparatus and assess the total protein transferred (see "Methods"). Step 2: Plot Standard Curve for each Target. Probe the membrane with each antibody. Plot the relative density of each protein versus protein load to generate a standard curve. Step 3: Optimize Protein Load. Determine the optimal total protein load by choosing the protein load corresponding to the middle of the standard curve linear range for each target. This offers the best chance of achieving target-specific quantitative data from the individual samples. (B) Quantification of Target Expression. Step 1: Data Acquisition and Quality Control. Assure that the total protein loaded per lane is within the linear dynamic range of the target protein for accurate normalization and sample quality verification. Step 2: Data Analysis. Use a similar methodology as qPCR40 to work up the data from western blotting to assure the results reflect the tested experimental conditions. The blot images from this figure are strictly to depict the western blot workflow, associated quality controls and data analysis steps with contrived data to clarify the data analysis steps.
