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SASBI - Bradford Assay

Protein quantification assay - Bradford Assay

References | Quick Guide | Discussion | Reagents | Procedure | Notes

References

  1. Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72: 248-254.
  2. Compton SJ, Jones CG (1985). Mechanism of dye response and interference in the Bradford protein assay. Anal Biochem, 151: 369-374.
  3. Read SM, Northcote DH (1981). Minimization of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Anal Biochem, 116: 53-64.
  4. Zor T, Selinger Z (1996). Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem, 236: 302-308.
  5. UNL classroom server Biochemistry protocols

Quick Guide

How does it work?

  • Absorbance shift in Coomassie Brilliant Blue G-250 (CBBG) when bound to arginine and aromatic residues
  • The anionic (bound form) has absorbance maximum at 595 nm whereas the cationic form (unbound form) has and absorbance maximum at 470 nm

Detection Limitations

  • 1-20 μg (micro assay) 20-200 μg (macro assay)

Advantages

  • Fast and inexpensive
  • Highly specific for protein
  • Very sensitive
  • Compatible with a wide range of substances
  • Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin)
  • Dye reagent is complex is stable for approximately one hour
  • The Zor and Selinger modified procedure can be performed in a microtiter plate

Disadvantages

  • Absorbance spectra of the two Coomassie Brilliant Blue G-250 species partially overlap making the standard curve very important
  • Non-linear standard curve over wide ranges
  • Response to different proteins can vary widely, choice of standard is very important

General Considerations

  • The dye binds to quartz cuvettes so it is usually better to use glass or plastic cuvettes
  • The dye reagent is usually more convenient to purchase than to make, due to the use of phosphoric acid

Micro Assay Procedure

  • Warm up the spectrophotometer for 15 min before use.
  • Dilute samples with buffer to an estimated concentration of 1 to 20 micrograms/milliliter
  • Prepare standards containing a range of 1 to 20 micrograms protein (albumin or gamma globulin are recommended) to a volume of 200 μl (to a volume of 100 μl if you are adding 1 M NaOH)
  • Prepare unknowns to estimated amounts of 1 to 20 micrograms protein per tube to 200 μl (100 μl if you are using 1 M NaOH)
  • (Optional) Add 100 μl 1 M NaOH to each sample and vortex.
  • Add 800 μl dye reagent and incubate 5 min.
  • Measure the absorbance at 595 nm.

Macro Assay Procedure

  • Warm up the spectrophotometer for 15 min. before use.
  • Dilute samples with buffer to an estimated concentration of 20 to 200 micrograms/ml
  • Prepare standards containing a range of 20 to 200 micrograms protein (albumin or gamma globulin are recommended) to a standard volume (generally 1 ml or less). See how to prepare and use a protein standard curve for suggestions as to setting up the standards.
  • Prepare unknowns to estimated amounts of 20 to 200 micrograms protein per tube, same volume as the unknowns.
  • (Optional) Add 0.25 ml 1 M NaOH to each sample and vortex.
  • Add 5 ml dye reagent and incubate 5 min.
  • Measure the absorbance at 595 nm.

Discussion

Bradford assay is a simple, rapid, inexpensive, sensitive and reproducible assay for quantification of proteins. It works by the action of Coomassie brilliant blue G-250 dye (CBBG), which binds specific amino acid residues exposed on the surface of the protein undergoing quantification. The dye molecule contains six phenyl groups and two sulfonic acid groups and so interactions are mainly weak and noncovalent and occurs via hydrophobic (Tryptophan, Tyrosine, Histidine and Phenylalanine) and electrostatic (Arginine, Lysine) residues. It is unaffected by many common laboratory chemicals (e.g. sucrose, urea, glycerol) but gives erroneously high absorbance readings when high levels of detergents (e.g. > 0.1% SDS and > 1% Triton series) or cellular-derived lipids, basic buffers and flavenoids are present in the sample. Excess detergent interacts hydrophobically with the dye and causes a wavelength shift and an overestimation of protein.

The Coomassie Brilliant Blue G-250 dye exists in three forms: red (» max = 470 nm), blue (» max = 590 nm) and green (» max = 650 nm). CBBG binds to amino acid residues in an anionic form, with the absorbance maximum at 595 nm (blue); on the other hand, the free dye in the solution is in the cationic form which has an absorbance maximum at 465 nm (red). The original Bradford assay is therefore based on the absorbance maximum (» max ) shift from 465 nm (cationic form) to 595 nm (anionic form) of the dye molecules in the presence of protein; and it is monitored at 595 nm in a spectrophotometer, and thus measures the CBBG complex with the protein.

CBB G-250

Chemical Structure of Coommassie Brilliant Blue G-250

It should be emphasized that the absorption spectra of the two forms of the dye overlap. This causes the assay to respond non-linearly in the standard curve, particularly toward the high end of its useful protein concentration range. All kit providers of the Bradford assay, and many researchers insist that the assay performs linearly. However, when a standard curve is performed it is noted that a second order curve will fit much better than a linear curve. The assay does perform linearly over short concentration stretches, and this has most likely resulted in the overall conclusion that the assay is linear. Bradford himself even notes that the assay is not linear over the whole range in the original article.

Various modifications to the original procedure have attempted to minimise variability in colour development with different protein samples and to increase the sensitivity of the assay, by replacing the Coomassie brilliant blue G–250 with an increased amount of Serva Blue G dye (Read and Northcote, 1981). Another modification of the procedure involved measuring the ratio of A590/A450. This effectively linearises the curve, and increases the sensitivity of the assay to 50 ng – 40 μg, even in the presence of up to 35–fold excess SDS detergent (Zor & Selinger, 1996).

Typical Bradford Assay Standard Curve

Typical Bradford Assay Standard Curve

One crucial part of this assay is the buffer blank. Since the assay responds non-linearly it is highly important to lock down the zero point. Because this point is so important to the curve fit, it is highly recommended that at least two buffer blanks be performed. If it is determined that interference is not occurring, as is the usual case, you can proceed to use a water buffer blank.

The choice of standard for this assay is very crucial to the success of the assay. Many investigators have noted abnormalities of using various standards with the Bradford assay. BSA was the original standard of choice, and is standard you are most likely to receive with the assay if purchased as a kit. However, it has been noted that BSA has a double than "normal" response in the assay and may not always be suitable. Several researchers therefore use Imunoglobulin G (IgG) as the preferred standard for the assay, or ovalbumin. If your protein of interest is high in arginine, you may need to find an Arg-rich standard protein as well, since this assay reponds primarily to Arg residues (8x as much as the other listed residues).

The CBBG dye used in the assay binds to quartz cuvettes quite strongly. Therefore, glass or plastic cuvettes should be utilized. Since this assay has a general tendency to bind to cuvettes, it is highly recommended to use disposable plastic cuvettes. This is by no means critical to the assay, but it does make cleaning the cuvettes much more convenient.

There are two major formats of this assay each with a different detection range. The micro assay format is designed for protein concentrations between 1-20 micrograms and the macro assay is designed for protein concentrations in the range of 20-100 micrograms. It is generally more convenient to use the micro format and dilute your protein down to the concentration range. The micro assay results in less chemical waste and allows the concentrate to last much longer. The assay can also be performed in a microwell plate, which is a very convenient way to process a large number of samples rapidly.

One consideration is to use a small amount of sodium hydroxide in the assay to help solubulize your protein. Some proteins, especially hydrophobic, membrane or "sticky" proteins tend to precipitate in the presence of coomassie dyes. If you observe a precipitate forming when you add the dye reagent to your sample try adding the specified amount of sodium hydroxide.

Reagents

Bradford Dye reagent: 0.05g of Serva Blue G dye (CBBG) is dissolved in 50 ml of an 88% (v/v) phosphoric acid solution and 23.5 ml absolute ethanol. The solution is made up to 500 ml with dd.H2O and stirred for 30 min at room temperature (RT) on a magnetic stirrer. The solution is then filtered through Whatman No. 1 filter paper and stored in an amber bottle at room temperature. Before use, the solution is checked for dye precipitation that would otherwise lead to erroneous protein quantification, and refiltered and recalibrated if necessary.

Standard BSA solution (1 mg/ml): 0.1 g BSA is dissolved and made up to 100 ml with PBS.

(Immunoglobulin G (IgG) or ovalbumin can be used as a standard solution in some cases)

Bradford assay procedure

  1. Construct a standard curve using the standard BSA solution (1 mg/ml) for the range of 0-50 μg. Determine linear regression equation (y = mx + C) and correlation coefficient (r2).
  2. Protein samples are made up to 50 μl with dd.H2O in 1.5 ml eppendorf microcentrifuge tubes
  3. 950 μl of Bradford reagent is then added. The tubes are sealed and gently vortexed to ensure even mixing of the dye reagent
  4. The reaction is allowed to continue for 10 minutes before absorbances are read at 595 nm wavelength (warm up the spectrophotometer 15 minutes before use).
  5. The conventional Bradford assay recommends the use of dye reagent as the blank, but water can also be sufficiently used; this is particularly viable because more than one form of Coomassie dye is monitored in the assay and the absorbances are relative to dd.H2O at each wavelength.
  6. Protein concentration estimations are calculated from the equations generated by linear regression analysis of standard curve for each batch of dye reagent made up.   

Notes

  • CBBG dye binds to quartz cuvettes quite strongly; therefore, glass or plastic cuvettes should be used.
  • It should be noted that the assay primarily responds to arginine residues (eight times as much as the other listed residues) so if you have an arginine rich protein, you may need to find a standard that is arginine rich as well.
  • Hydrophobic, membrane or sticky proteins tend to precipitate in the presence of Coomassie dyes; it is therefore recommended that small amount of sodium hydroxide is used in the assay to help in their solubility. (In this case add equal volumes of the sample and 1 M NaOH, and make up to 1000 μl with the dye reagent) Reference: UNL Classroom server Biochemistry protocols
  • Recent investigations have shown non-linearity at 595 nm since there is an overlap of the red and blue dye species at this wavelength. To overcome this problem, monitoring the decrease in the [blue dye] free as well as the decrease in absorbance of the red form by establishing the A590/A450 ratio can be adopted where. The ratio exhibits linearity from 20 ng to 40 μg protein. This recent modification is reported to improve sensitivity ten-fold and allows quantification in the presence of up to 35-fold excess detergent.
  • General notes

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