SBRU

Biochemistry and Assays

BCA assay

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Content from: [bibliplug id=1623]

Quick Guide

How does it work?

  • The first step is a Biuret reaction which reduces Cu+2 to Cu+1
  • In the second step BCA forms a complex with Cu+1 which it purple colored and is detectable at 562 nm

Detection Limitations

  • 0.2 — 50 μg

Advantages

  • Less susceptible to interference from common buffer substances
  • Very sensitive and rapid if you use elevated temperatures
  • Compatible with many detergents
  • Working reagent is stable
  • Very little variation in response between different proteins
  • Broad linear working range

Disadvantages

  • The reaction does not go to completion when performed at room temperature or 37°C. This can be a problem if you are assaying a large number of proteins
  • Dilution is often necessary for concentrated protein samples

General Considerations

  • This method is related to the Lowry method. However, the use of BCA instead of the Folin-Ciocalteu reagent, makes it less susceptible to interference
  • Since reduced copper is detected in the procedure, make sure that the distilled water used in the procedure is fed from plastic lines and not copper lines. In general water from 18 megaohm water polishers is satisfactory

Procedure

Bicinchoninic Acid Solution

Commercially available (e.g. Sigma B 9643). Contains BCA, sodium carbonate, sodium tartrate and sodium bicarbonate in 0.1 M NaOH (pH 11.25)

Copper Sulfate Solution

4% CuSO4*5H2O

  • Prepare the required amount of protein determination reagent by adding 1 volume copper sulfate solution to 50 volumes of bicinchoninic acid solution.
  • Set up test tubes containing samples and known amounts of bovine serum albumin in the range of 0 to 100 micrograms. Each tube should contain 0.1 mL total volume.
  • Add 2.0 mL of the protein determination reagent to each tube and vortex.
  • Incubate the tubes at 37°C for 30 min (Alternatively 2h at room temperature, or 15 minutes at 60°C).
  • Cool the tubes to room temperature and determine the absorbance at 562 nm. After cooling to room temperature, the blank continues to increase in absorbance at approximately 2.3% per 10 min (if you use 60°C incubation temperature, the absorbance will not increase appreciably). Because of the small volume in each tube, cuvettes with an approximate 1 mL capacity should be used for the absorbance measurements.

Discussion

This assay measures the formation of Cu+1 from Cu+2 by the Biuret complex in alkaline solutions of protein. It was originally thought that the mechanism of the assay was the same as in the Lowry assay, but it has since been determined that there are two distinct reactions that take place with copper ions unique to the BCA assay. The first reaction occurs at lower temperatures and is the result of the interaction of copper and BCA with the following residues: cysteine, cystine, tryptophan and tyrosine. At elevated temperatures it has been shown that the peptide bond itself is responsible for color development. This is why performing the assay at 37°C or 60°C increases the sensitivity and reduces the variation in the response of the assay to protein composition.

Chemical Structure of BCA
Chemical Structure of BCA

The BCA reagent replaces the Folin-Ciocalteu reagent used in the Lowry assay with bicinchoninic acid. The BCA reagent forms a complex with Cu+1, which has a strong absorbance at 562 nm. BCA is advantageous in that it does not interact with as many substances as the Folin-Ciocalteu reagent, especially detergents and buffers. The BCA assay is limited in that it interacts with most reducing agents and copper chelators. In general, these are not critical components of buffers and can be easily eliminated prior to assay.

Typical Standard Curve for a BCA Assay
Typical Standard Curve for a BCA Assay

References

[bibliplug keywords=bca order_by=’last_name, year-, title’]

Ultraviolet absorbance of aromatic amino acids

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Content from: [bibliplug id=1625]

Quick Guide

How does it work?

  • Monitors the absorbance of aromatic amino acids, tyrosine and tryptophan or if the wavelength is lowered, the absorbance of the peptide bond. Higher order structure in the proteins will influence the absorption

Detection Limitations

  • 20 μg to 3 mg

Advantages

  • Quick
  • Sample can be recovered
  • Useful for estimation of protein before using a more accurate method
  • Well suited for identifying protein in column fractions

Disadvantages

  • Highly susceptible to contamination by buffers, biological materials and salts
  • Protein amino acid composition is extremely important, thus the choice of a standard is very difficult, especially for purified proteins
  • Absorbance is heavily influence by pH and ionic strength of the solution.

General Considerations

  • This is often used to estimate protein concentration prior to a more sensitive method so the protein can be diluted to the correct range

Procedure

Quantitative Procedure

  • Zero the spectrophotometer with a buffer blank
  • Make a standard curve using your standard of choice in the expected concentration range, using the same buffer that your unknown sample is in.
  • Take the absorbance values at 280 nm in a quartz cuvette
  • Place sample into quartz cuvette (make sure concentration is in the range of 20 μg to 3 mg
  • Take absorbance at 280 nm

Estimation Procedure

  • Zero spectrophotometer to water (or buffer)
  • Take the absorbance at 280 nm in a quartz cuvette
  • Change wavelength to 260 nm and zero with water (or buffer)
  • Take absorption at 260 nm in a quartz cuvette
  • Use the following equation to estimate the protein concentration
  • [Protein] (mg/mL) = 1.55*A280 – 0.76*A260

Discussion

Determination of protein concentration by ultraviolet absorption (260 to 280 nm) depends on the presence of aromatic amino acids in proteins. Tyrosine and tryptophan absorb at approximately 280 nm. Higher orders of protein structure also may absorb UV light or modify the molar absorptivities of tyrosine and tryptophan and thus the UV detection is highly sensitive to pH and ionic strength at which measurement is taken. Many other cellular components, and particularly nucleic acids, also absorb UV light. The ratio of A280/A260 is often used as a criterion of the purity of protein or nucleic acid samples during their purification. The real advantages of this method of determining protein concentration are that the sample is not destroyed and that it is very rapid. Although different proteins will have different amino acid compositions and thus different molar absorptivities, this method can be very accurate when comparing different solutions of the same protein.

To make an accurate determination of protein concentration, you will have to produce a standard curve (A280) with known amounts of purified protein. You will also have to provide a blank that is appropriate for the sample and contains the same concentrations of buffer and salts as the sample. It is often convenient to dialyze the sample and measure the absorbance of the retentate (still in the dialysis sack) using the dialysate as the blank. Care must be taken to use quartz cuvettes, since glass absorbs UV light. A handy equation to estimate protein concentration that is often used is

[Protein] (mg/mL) = 1.55*A280 – 0.76*A260

However, it is also a good idea to always use a standard curve and suggested that you evaluate the agreement of the results using the above equation with results using a standard curve.

This method is the least sensitive of the methods discussed here. For increased sensitivity, the wavelength can be lowered to the range of 210 to 225 nm. This measures the amide bond in proteins. However it is much more subject to interference from many more biological components and compounds used to make buffer solutions.

If you don’t know what the protein concentration of an unknown sample is likely to be, the ultraviolet method might be a good starting point. Prepare a standard curve for the absorbance at 280 and 260 nm. After you have the data for the standard curve, rezero the spectrophotometer with water. Place your samples into a dry 1 mL quartz cuvette and read the absorbance. If the A280 of your unknown sample is less than 2, you should probably not dilute your sample further. If the absorbance is <2, dilution will be required. When you are finished with the first measurement, the unknown can be returned to its original tube with minimal loss.

References

[bibliplug keywords=’uv’ order_by=’last_name, year-, title’ category=’resources’]