Quantitative Western Blot Overview
Quantitative Western Blot workflows provide analysis capabilities for comprehensive Western blot projects that start with validation Experiments and lead to Target Analysis Experiments that include fold change calculations and statistical calculations.
Summary of Differences between Qualitative and Quantitative Western Blot Experiments
This table highlights some key differences between Qualitative and Quantitative Western Blot Experiments.
| Qualitative Western Blot | Quantitative Western Blot | |
|---|---|---|
| Antibody Validation | ||
| Linear Range Determination | ||
| Housekeeping Protein Validation | ||
| Warnings on Target Analysis Starting Pages for Incomplete Validation Experiments | ||
| Set up calculations step in Target Analysis (used to setup fold change calculations, etc.) |
Overview of Quantitative Western Blot Experiments
Antibody Validation Experiment
| Experiment Type | Purpose | Process | Detection | Maximum Images | Maximum Targets |
|---|---|---|---|---|---|
| Antibody Validation | Validate primary antibodies for use in your intended assay and sample context. | Evaluate antibodies in your assay to confirm expected performance. | Fluorescence, chemiluminescence | 3 | 4 |
Linear Range Determination Experiment Types
See Finding the Combined Linear Range with Empiria Studio for more information about why the linear range needs to be determined and how linearity and proportionality are determined in Empiria Studio.
| Experiment Type | Purpose | Process | Detection | Maximum Images | Maximum Targets |
|---|---|---|---|---|---|
| Linear Range: TPS + Targets | Find the range of sample loading that produces a linear, proportional change in signal intensity for both the target and total protein stain (TPS). This is the combined linear range. | Quantify the total protein and target signals in each lane, using serial dilutions of your sample. Empiria Studio will plot the results to help you identify areas of overlap (the combined linear range). You can then determine an appropriate range of sample loading for accurate detection of both the target and TPS. | Fluorescence, chemiluminescence | 3 | 4 |
| Linear Range: ILC + Targets | Find the range of sample loading that produces a linear, proportional change in signal intensity for both the target and internal loading control (ILC). This is the combined linear range. | Quantify the ILC and target signals in each lane, using serial dilutions of your sample. Empiria Studio will plot the results to help you identify areas of overlap (the combined linear range). You can then determine the appropriate range of sample loading for accurate detection of both the target and ILC. | Fluorescence, chemiluminescence | 3 | 4 |
HKP Validation
| Experiment Type | Purpose | Process | Detection | Maximum Images | Maximum Targets |
|---|---|---|---|---|---|
| HKP + Total Protein Validation | Determine if your chosen housekeeping protein (HKP) is stably expressed in your samples and conditions. | Analyze blot images that were scanned separately. | Fluorescence, chemiluminescence | 3 | 4 |
| HKP Validation | Observe protein expression stability across samples and conditions. If expression of a protein varies across samples, that protein is most likely not suitable for use as an internal loading control in Western blot normalization. | Quantify and compare signal from protein bands in sample lanes. | Fluorescence, chemiluminescence | 3 | 4 |
Target Analysis
| Experiment Type | Purpose | Process | Detection | Maximum Images | Maximum Targets |
|---|---|---|---|---|---|
| Analysis: TPS + Targets | Analyze your target bands, using total protein staining (TPS) for normalization. | Quantify the total protein and target signals on your blot. Empiria Studio will automatically normalize the target results, using TPS to correct for lane-to-lane variation. You can view the results and export images and data for this Analysis. | Fluorescence, chemiluminescence | 3 | 4 |
| Analysis: HKP + Targets | Analyze your target bands, using your HKP for normalization. | Quantify the HKP and target signals on your blot. Empiria Studio will automatically normalize the target results, using the HKP to correct for lane-to-lane variation. You can view the results and export images and data for this Analysis. | Fluorescence, chemiluminescence | 3 | 4 |
| Analysis: Pan-Target + PTM-Targets | Analyze your modified target protein, using the pan-protein for normalization. | Quantify the pan-target and PTM-target signals on your blot. Empiria Studio will automatically normalize the PTM-target results, using the pan-target to correct for lane-to-lane variation. You can view the results and export images and data for this Analysis. | Fluorescence, chemiluminescence | 3 | 4 |