Struggling to get reliable results from your Bradford protein assay? You're not alone. From inconsistent standards to unexpected interference, even small mistakes can throw off your data. In this guide, we’ll walk through the most common issues and how to troubleshoot them, plus, we share how smarter lab procurement strategies can help reduce errors before they start.
About Bradford assays
The Bradford protein assay is an easy and simple method for quantification of your protein concentration, yet still requires occasional troubleshooting.
The Coomassie Brilliant Blue dye binds to basic and aromatic amino acid residues, causing a color change from brown to blue, and an absorbance shift. Protein concentration can be determined by referencing a standard protein, most commonly BSA (Bovine serum albumin).
Variables such as temperature, wavelength, detergents, and even cuvette types can impact measurements. Below are common problems and troubleshooting tips to help ensure accurate, reliable results.
Troubleshooting low absorbance in Bradford assay samples
- Possible Cause: Low molecular weight.
Solution: The Bradford assay detection limit is ~3,000-5,000 Daltons. Use an alternative assay (e.g., BCA) for smaller proteins. - Possible Cause: Interfering substances.
Solution: The sample may contain interfering substances, such as detergents (Table 1). Dilute your sample and ensure standards are prepared in the same buffer.
What to do when Bradford assay absorbance is too high
- Possible Cause: Protein concentration is too high.
Solution: Dilute your sample and repeat the assay.
- Possible Cause: Interfering substances.
Solution: Same as above, dilute and ensure consistency with standard buffer.
Bradford assay standards showing low absorbance? Try these fixes
- Possible Cause: Old or improperly stored dye reagents.
Solution: Replace outdated Bradford reagent (expires ~12 months); store at 4°C. - Possible Cause: Incorrect standard dilutions.
Solution: Follow the manufacturer's protocol precisely for creating your protein standard dilutions. - Possible Cause: Bradford reagent may be too cold.
Solution: Bring to room temperature before use. - Possible Cause: Absorbance measured at incorrect wavelength.
Solution: Measure the absorbance at 595 nm.
Scientists save 6–10 hours per week by ordering lab supplies with ZAGENO.
Dark blue Bradford assay samples? Here’s what it means
- Possible Cause: High alkaline concentrations.
Solution: Alkalinity may raise pH beyond assay limits. Dilute or dialyze the sample.
Why your Bradford assay sample has precipitates
- Possible Cause: Detergents in your protein buffer.
Solution: Dialyze or dilute the sample to reduce detergent concentration.
How to handle interfering substances in Bradford assays
One of the most common issues when performing a Bradford assay is the presence of interfering substances in the buffer. Table 1 below summarizes a list of the most commonly used compatible substances and their concentrations. For detailed information, please refer to the manufacturers’ protocol.
Note: This is not an exhaustive list. Some substances may also alter pH, further impacting the assay.
Table 1: Compatible Substance Concentrations in the Bradford Assay (Modified from Bio-Rad Quick Start #4110065 and Tech Tip #68)
If your buffer contains incompatible substances:
- Possible Cause: High concentration of an interfering substance.
Solution: Dilute the sample to the point of no interference if your protein concentration is high enough, or remove the substance via dialysis. - Possible Cause: Substance not listed in compatibility table.
Solution: Run two standard curves (in water and buffer). Plot a graph of protein concentration versus absorbance. If slopes match, the buffer is not interfering.
Bradford assay best practices: Quick technical tips
- The dye can react with quartz cuvettes; use glass or plastic cuvettes instead,
- Ensure glassware is clean before use to prevent contamination.
- Perform assays with Bradford reagent at room temperature.
Improve reproducibility with better lab procurement
Reliable assays start with consistent supplies. ZAGENO helps labs reduce variability with easy access to over 40 million scientific products from over 5,300 trusted suppliers, all in one place.
Want to prevent future assay issues? Better procurement is a great place to start.
