Figure 4

Western blotting data analysis workflow (example using contrived data). The data analysis workflow can be tabulated in EXCEL with formula propagation (see supplemental WB Data Analysis EXCEL Spreadsheet). The columns are partitioned to incorporate the critical data components and calculations: (1) Gel number. (2) Target: Interblot Control (IBC): The IBC is recommended to be an equalized pooled sample from the positive experimental samples from each biological group. LC: Loading Control: The loading control used for normalization. Either the lane density of total protein or a specific loading control protein that does not change in expression between biological groups. T1: The target protein tested for expression differences between biological groups. (3) BioGroup_Sample: The individual samples within each biological group (i.e.: the tested treatment/condition). (4) Band Density: The density/volume of a given protein band as revealed with an imaging system from a fluorescence or chemiluminescence signal. (5) Normalized Ratio (NR): The density ratio of the target protein versus the loading control within each gel lane (sample). (6) Relative Normalized (RNR): The NR per sample divided by the average NR for the untreated (control) samples within each gel. (7) Relative Normalized RNR: The RNR per sample divided by the average RNR for untreated (control) samples spanning a given experiment (between gels). (8) Interblot Control (IBC) Ratio (IBCR): NR per sample divided by the NR of the IBC within each gel (Controls for any inter gel/blot variability in sample loading, transfer efficiency and other sources of experimental variability when running multiple gels for the same target). The IBCR is an essential control when the number of samples for a given experiment exceed the number of available wells of a single gel. (9) Relative Normalized IBCR: The IBCR per sample divided by the average IBCR for untreated (control) samples spanning a given experiment (between gels). (10) Relative Normalized NR: The NR per sample divided by the average NR for untreated (control) samples spanning a given experiment (between gels). The charts demonstrate the statistical significance between the same set of treated and untreated samples based on the different calculation methodologies. ****p-value < 0.0001, ns non-significant.