Estimation of protein concentration

B.5 Estimation of protein concentration

B.5.1 Absorption of UV light

Proteins containing tryptophan, tyrosine or phenylalanine residues absorb UV light at 280 nm in a concentration-dependent manner. Consequently, a spectrophotometer may be used to determine the increased absorption obtained when a protein is dissolved in a buffer (the buffer alone is used to ‘zero’ the spectrophotometer, so this ‘difference’ is obtained directly) and this value used: (i) to calculate the concentration of a pure solution using a published extinction coefficient (E 1% 1 cm athe absorbance at 280 nm due to a 10 mg/ml solution measured in a quartz cuvette with a 1-cm light path; values for commonly used proteins are given in Table B.1); or (ii) if the solution contains more than one protein, to estimate their total concentration.

MATERIALS AND EQUIPMENT Protein solution, of unknown concentration Buffer solution, used for dissolving the protein Spectrophotometer, capable of operating at UV wavelengths Quartz cuvettes, typically with 1.0- or 0.5-cm light path

METHOD

1 Turn on the spectrophotometer and allow it to stabilize for 15 min at 280 nm, in the absorbance rather than transmission mode.

2 Add buffer to the cuvette and use it to adjust the absorbance to zero.

3 Replace the buffer with protein solution (or use another cuvette if you have a matched set) and read the absorbance.

4 Using its extinction coefficient calculate the concentration of protein, remembering to allow for any dilutions made, and standardize for a 1-cm cuvette.

A P P E N D I X B: Basic techniques and useful data 365

Table B.1 Molecular weights and spectral properties of immunoglobulins and antigens of immunological interest

Wavelength Protein

0.2 m NaCl pH 7.5 IgA

0.2 m NaCl pH 7.5 IgD

5 m guanidine HCl γ chain

0.01 n HCl light chain

0.01 n HCl Fab γ

F(ab γ ) 2 104 000

PBS pFc′ γ

PBS Ovalbumin

PBS Human serum albumin

PBS Bovine serum albumin

Water Fowl γ-globulin

– Keyhole limpet haemocyanin

3 000 000 (Megathura crenulata)

Squid haemocyanin 611 800 (Ommatostrephes sloani pacificus) Murex haemocyanin

Water Limulus haemocyanin

Water 2,4-dinitrophenyl (DNP)

0.5 m phosphate pH 7.4 4-hydroxy-3-nitro-5-iodo phenacetyl azide (NIP azide)

0.15 m NaCl, p.02 m Κ phosphate pH 7.4

TECHNICAL NOTES • To ensure that the relationship between concentration and UV absorption is linear, the

absorbance should be below 2.0, and ideally between 0.1 and 1.5 absorbance units. • If the extinction coefficient of the protein is not known, or if the solution is a mixture of several proteins, total protein concentration may be calculated according to the following equation:

Protein concentration, mg/ml = 1.55 × A 280 −

0.77 × A 260

where A 280 is absorbance value at 280 nm and A 260 is absorbance value at 260 nm. • Protein solutions frequently show much greater absorbance at wavelengths below 280 nm; however, so do many other materials. The ratio of the A 280 and A 260 readings should be below

0.6 for protein solutions; higher ratios usually indicate contaminants such as nucleic acids, pep- tides, detergents and preservatives such as sodium azide. If you know this to be a problem, use

a colorimetric method. • The technique is reasonably accurate for protein solutions at concentrations greater than

0.1 mg/ml.

A P P E N D I X B: Basic techniques and useful data

B.5.2 Lowry technique

The estimation of protein concentration by UV absorption may be altered by the presence of cer- tain detergents and buffers that absorb light strongly in these wavelengths. This problem is over- come by selecting chemicals with low UV absorption, e.g. substitution of the non-ionic detergent Renex 30 for Nonidet P-40. More often, however, it is necessary to use a different principle for estimation of protein content; for example, the colorimetric method developed by Lowry.

The Lowry technique involves the construction of a standard curve of colour versus protein concentration by reacting different concentrations of a known protein with Folin and Ciocalteu’s phenol reagent (bovine serum albumin as a ubiquitous standard). The blue colour generated by the unknown protein solution can then be converted into concentration units by reference to the standard curve. Accuracy is enhanced if the known and unknown proteins are structurally related.

Preparation in advance

MATERIALS AND EQUIPMENT As Section B.5.2, but in addition:

Protein for use as standard Phosphate-buffered saline (PBS)

METHOD

1 Dissolve the standard protein in PBS to 1 mg/ml.

2 Centrifuge or filter to remove any undissolved material.

3 Determine the 280 nm absorbance of the protein solution and calculate its precise concentration using the extinction coefficient.

4 Dispense in small aliquots and store at –20°C for use.

Estimation of unknown protein solution

MATERIALS AND EQUIPMENT Standard and unknown protein solutions Sodium carbonate, 2.0% w/v in 0.1 M sodium hydroxide Cupric sulphate, 1.0% w/v in distilled water Sodium potassium tartrate, 2.0% w/v in distilled water Folin and Ciocalteu’s reagent Spectrophotometer, visible light

METHOD

1 Prepare a dilution series of the standard protein solution in five steps between 0 and 500 µg/ml in 100 µl (final volume).

2 Prepare a series of dilutions of the unknown protein solution so that at least one tube falls within the range of the standard series. (As a first approximation, prepare a 1 : 5 dilution series through three steps and use 100 µl of each.) Include also a tube containing only the buffer used to dissolve the unknown protein, if this differs from that used to dissolve the protein standard.

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A P P E N D I X B: Basic techniques and useful data 367

3 Mix an equal volume of copper sulphate and sodium potassium tartrate solutions, remove

1 ml and mix with 50 ml sodium carbonate solution (this mixture must be freshly prepared for each assay). Add 1 ml of this final mixture to each of the tubes containing standard or unknown protein solutions.

4 Add 100 µl of Folin and Ciocalteu’s reagent to each tube and mix vigorously.

5 Incubate the tubes at room temperature for 15 min and quantify the colour reaction in a spectrophotometer at 650 nm.

6 Plot absorbance against protein concentration for the standard solution (the curve deviates slightly from linearity) and from this determine the protein concentration equivalent to the colour reaction of the unknown.

TECHNICAL NOTES • As a guide, a protein solution of 250 µg/ml initial concentration yields a colour reaction with an

absorbance of approximately 0.4. The lower limits of detection are about 5 µg/ml. • If the buffer used to prepare the unknown solution gives a colour reaction in the absence of protein, this value should be subtracted from the absorbance of the unknown solution. In addition, it is not uncommon to find that buffer molecules or non-ionic detergents react with the phenol reagent to form a precipitate, without affecting the validity of the colour reaction in the supernatant.

• If a blue reaction is seen in the tube containing only the buffer used to prepare the standard curve, this indicates protein contamination, usually of the phenol reagent. • The cupric sulphate and sodium potassium tartrate should be dissolved independently before mixing, to avoid precipitation.

B.5.3 BCA protocol for protein estimation

This is a colorimetric method for measurement of total protein in aqueous solution. It utilizes bicinchroninic acid (BCA) and the reduction of copper ions (Cu 2+ → Cu + ) in the presence of pro- tein to produce a colour change which is read at an absorbance of 562 nm. • BCA reagent is commercially prepared by and available from Pierce & Warriner Ltd.

B.5.4 Bradford method for total protein estimations

This is a colorimetric assay, but uses Coomassie brilliant blue G-250. When this reagent binds to proteins at an acidic pH there is an absorbance shift, and the colour change is read spectrophoto- metrically at 595 nm. As above, a commercially prepared reagent is available from Pierce & Warriner Ltd.