Description
Bradford Reagent (1X, Ready-to-Use) – 2 mL
A ready-to-use Coomassie Brilliant Blue G-250 reagent for fast, sensitive colorimetric quantification of total protein in cell lysates, tissue extracts, purified proteins and column fractions. Add, wait five minutes, read at 595 nm – no heating, no reagent preparation.
Key features
- Ready to use – supplied as a 1X working solution; no dilution, filtration or mixing of components required.
- Fast – colour develops in 5 minutes at room temperature and is stable for up to 60 minutes.
- Sensitive – detects protein from approximately 1 µg/mL (micro assay) up to 1,500 µg/mL (standard assay).
- Flexible – compatible with 96-well microplate, 1 mL cuvette and micro-cuvette formats.
- Tolerant of reducing agents – unaffected by DTT, β-mercaptoethanol, EDTA and most common buffer salts.
- Trial size – 2 mL is ideal for evaluating the reagent, teaching labs, or a small pilot experiment before scaling up.
What is the Bradford assay?
The Bradford assay is a dye-binding method for measuring total protein concentration, first described by Marion Bradford in 1976. It remains one of the most widely used protein assays in research laboratories because it is quick, inexpensive and requires only a spectrophotometer or plate reader.
The reagent contains the dye Coomassie Brilliant Blue G-250 in an acidic solution. Under these conditions the free dye exists predominantly in its red-brown cationic form, which absorbs maximally at 465 nm. When the dye binds to protein it is stabilised in its blue anionic form, shifting the absorbance maximum to 595 nm. The increase in absorbance at 595 nm is directly proportional to the amount of protein present, so an unknown sample can be quantified by comparison with a standard curve of known protein concentrations.
Dye binding occurs mainly through interactions with basic amino acid residues (arginine, lysine and histidine) and, to a lesser extent, aromatic residues. Because the response depends on amino acid composition, results are best expressed relative to a standard such as bovine serum albumin (BSA) or bovine gamma globulin (BGG).
How it works
Sample or standard is combined with Bradford reagent in a microplate well or cuvette.
Coomassie G-250 binds to protein and converts from its red-brown to its blue form.
Absorbance is measured at 595 nm after 5 minutes at room temperature.
Protein concentration is interpolated from a BSA or BGG standard curve.
Specifications
| Product | Bradford Reagent, 1X ready-to-use |
|---|---|
| Volume | 2 mL |
| Format | Liquid; single vial. Sufficient for approximately 10 microplate wells (200 µL reagent per well) or 2 standard cuvette assays (1 mL reagent). |
| Dye | Coomassie Brilliant Blue G-250 in phosphoric acid / methanol |
| Detection wavelength | 595 nm (acceptable range 570–610 nm) |
| Standard assay range | 100–1,500 µg/mL |
| Micro assay range | 1–25 µg/mL |
| Incubation | 5 minutes at room temperature; read within 60 minutes |
| Recommended standard | BSA or bovine gamma globulin (BGG), 0–2 mg/mL |
| Storage | 2–8 °C, protected from light. Do not freeze. |
| Shelf life | 12 months from date of receipt when stored as directed |
| Shipping | Ambient or blue ice |
How to use – assay protocol
Allow the reagent to reach room temperature and invert gently several times before use. Do not shake vigorously, as foaming will interfere with pipetting. Prepare a fresh set of protein standards (for example BSA at 0, 125, 250, 500, 750, 1,000, 1,500 and 2,000 µg/mL) in the same buffer as your samples.
Microplate procedure (standard assay, 100–1,500 µg/mL)
- Pipette 5 µL of each standard or unknown sample into separate wells of a clear, flat-bottom 96-well plate. Run each in duplicate or triplicate.
- Add 200 µL of Bradford reagent to each well and mix by gently tapping the plate or with an orbital shaker for 30 seconds.
- Incubate for 5 minutes at room temperature.
- Measure absorbance at 595 nm within 60 minutes.
- Subtract the blank (buffer only) reading, plot A595 against standard concentration, and read unknowns from the curve.
Cuvette procedure (standard assay)
- Pipette 20 µL of standard or sample into a clean 1.5 mL disposable cuvette.
- Add 1 mL of Bradford reagent and mix by gentle inversion or pipetting.
- Incubate for 5 minutes at room temperature.
- Read absorbance at 595 nm against a reagent blank.
Micro assay (1–25 µg/mL)
- Mix equal volumes of sample and reagent (for example 150 µL of each in a microplate well, or 1 mL of each in a cuvette).
- Incubate for 5–10 minutes at room temperature and read at 595 nm.
- Prepare standards in the 1–25 µg/mL range for this format.
- The standard curve is non-linear at higher concentrations; fit a quadratic or four-parameter curve rather than a straight line, or restrict the working range.
- Dilute samples so that readings fall within the standard range; samples reading above the top standard should be diluted and re-assayed.
- Always prepare standards in the same buffer as the samples so that any buffer effects cancel out.
- Clean cuvettes immediately after use – bound Coomassie stains plastic and glass. Rinse with methanol or ethanol if needed.
Sample compatibility and interference
The Bradford assay is compatible with most common lysis and purification buffers, including Tris, HEPES, phosphate, sodium chloride up to 1 M, EDTA, glycerol, and reducing agents such as DTT and β-mercaptoethanol. The main sources of interference are detergents and strongly basic conditions.
| Substance | Compatibility |
|---|---|
| DTT, β-mercaptoethanol, TCEP | Compatible |
| EDTA, EGTA, glycerol, sucrose | Compatible |
| Tris, HEPES, PBS, NaCl (≤1 M) | Compatible |
| Urea (≤3 M), guanidine-HCl (≤3.5 M) | Compatible at reduced concentrations |
| SDS, Triton X-100, Tween-20, NP-40 | Interferes above ~0.1 % – keep detergent in samples and standards equal, or dilute |
| Strong bases (pH > 9), high-concentration NaOH | Interferes – neutralise before assay |
Applications
- Normalising protein loading for Western blot, SDS-PAGE and ELISA.
- Monitoring protein purification – quick screening of column fractions.
- Quantifying cell and tissue lysates before downstream assays.
- Determining concentration of purified recombinant proteins and antibodies.
- Teaching and training laboratories.
Storage and handling
Store at 2–8 °C protected from light and do not freeze. The reagent contains phosphoric acid and methanol: wear gloves and eye protection, avoid contact with skin, and dispose of waste according to local regulations. A small amount of dye precipitate may form on storage; this does not affect performance and the reagent can be inverted gently before use.
Frequently asked questions
What is the difference between the Bradford, BCA and Lowry assays?
Bradford is the fastest (5 minutes, no heating) and is tolerant of reducing agents, but is sensitive to detergents and shows more protein-to-protein variation. BCA tolerates detergents but is affected by reducing agents and requires a 30-minute incubation at 37 °C. Lowry is the oldest of the three and is the most affected by interfering substances.
Which protein standard should I use?
BSA is the most common choice. Bovine gamma globulin (BGG) gives a response closer to that of many mixed-protein samples such as cell lysates and serum. Whichever you choose, use it consistently across experiments you wish to compare.
Can I use this reagent with samples containing SDS?
Detergent concentrations above about 0.1 % raise the background and distort the standard curve. Either dilute the sample below this level, or add the same detergent concentration to your standards so the effect is matched.
Why does my standard curve flatten at high concentrations?
This is normal for the Bradford assay. The dye becomes limiting as protein increases. Use a non-linear curve fit or restrict your standards to the range where your samples fall.
How many assays can I run with 2 mL?
Approximately 10 microplate wells at 200 µL reagent per well, or 2 cuvette assays at 1 mL per cuvette. Larger pack sizes are available for routine use.
Need a larger volume?
This 2 mL trial size lets you validate the reagent in your own samples. Bradford reagent is also available in larger pack sizes for routine protein quantification – contact our team for bulk pricing.
