Unequal sample loading, in terms of the amount of protein per lane, can cause variation in signal intensities, which can lead to inconsistent results and inaccurate conclusions. To prevent loading variability from disrupting your data, it is important to measure the total protein concentration of each sample. Additionally, normalization with an internal loading control (e.g., housekeeping protein or total protein stain) can only account for small pipetting errors and not for different amounts of loaded protein.
Measuring Protein Concentration
There are three primary methods to determine protein concentration: Bradford, Lowry, and bicinchoninic acid (BCA) assays. Each has specific constraints that may impact your research, so consider them carefully before proceeding. Keep in mind that you can use the Concentration Panel in Image Studio™ Software to assign concentration values and determine unknown concentrations.
| Bradford Assay | Lowry Assay | BCA Assay | |
|---|---|---|---|
Chemistry | Coomassie blue dye binds to proteins, forming a dye-protein complex. The absorbance spectrum of the complex is different from unbound dye, allowing protein quantification at about 595 nm. | Copper ions are reduced by proteins in an alkaline medium. The Folin-Ciocalteu reagent reacts with the copper ions to form a blue-colored complex, allowing protein quantification at about 750 nm. | Copper ions chelate in the presence of protein. BCA detects reduced copper ions in the complex, forming a violet-colored complex and allowing protein quantification at about 562 nm. |
Sample Compatibility | Less compatible with commonly used sample preparation substances like detergents, lysing agents, etc. | Less compatible with commonly used sample preparation substances like detergents, lysing agents, etc. | More compatible with commonly used sample preparation substances like detergents, lysing agents, etc. |
Workflow | Short incubation at room-temperature | Longer incubation times | Longer incubation times |
Accuracy | Higher protein-to-protein variation; low accuracy | Higher protein-to-protein variation; high accuracy | Lower protein-to-protein variation; high accuracy |
Once you have estimated the protein concentration of each sample, a dilution series will then allow you to establish your optimal loading amount based on the linear range of your Western blot. Learn how to validate the linear range for your target proteins and internal loading controls with Determining the Linear Range for Quantitative Western Blot Detection. As always, ensure that equivalent amounts of protein are loaded in each lane for quantitative analysis, regardless of your protein concentration method.
Protein Concentration and Western Blotting
Estimating your protein concentration and choosing an assay can sometimes be difficult. Luckily, our Protein Concentration learning path in the Lambda U® Education Portal makes it much easier. Use promo code: protein-concentration to access this course for free, and please don't hesitate to contact us with any questions or concerns.
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