Proteins are the fundamental units for research from years, whether they are in the form of enzymes catalyzing thousands of reactions or drugs being discovered to treat diseases. Whatever it is characterization of proteins and their purification is required in each field and determination of the protein concentration is necessary for this. Many assays have been designed to measure protein concentrations which are sensitive and accurate. But there are many substances which interfere in the measurements and therefore each method has its advantages and disadvantages. Choosing an appropriate assay depends on our requirements and our sample. Main factors which influence are sensitivity, specificity, ease of use, cost and range of useful response.
Here we will discuss some of the main methods and their good and the bad side.
1. Absorbance at 280nm
Proteins absorb light in UV region mostly at 280nm due to presence of aromatic amino acids as tryptophan, tyrosine and phenylalanine. Every protein has different amino acids and their extinction coefficient vary accordingly. Thus absorbance at 280nm can be monitored easily and concentration is calculated if extinction coefficient is known.
The technique has advantages of being simple, rapid and recovery of the sample after measurement.
But it also has disadvantages like interference from chromogenic substances, even nucleic acid absorbs in this range, so there is a strict requirement of pure protein for accurate results. If only nucleic acids are the contaminants, we can still calculate protein concentration using the formula:
Protein (mg/ml) = 1.55 Ã- A280 - 0.76 Ã- A260
2. Lowry's method:
This method is based on biuret reaction, where Cu2+ binds to nitrogen of peptide bonds and coverted to Cu+ in alkaline conditions. It is followed by reaction with Folin-Ciocalteau reagent where phosphomolybdotungstate is reduced to heteropolymolybdenum blue due to oxidation of aromatic amino acids giving blue colour. The amount of protein is determined by measuring absorbance at 750 nm against a standard graph of some known protein. Most widely used standard protein is Bovine serum albumin (BSA).
The technique is sensitive up to 0.01mg/ml concentration of the protein although buffers like tris and ammonia can interfere in the measurements.
3. The Bicinchoninic Acid (BCA) Assay:
BCA reaction is also based on same biuret reaction showing conversion of Cu2+ to Cu+ under alkaline conditions, followed by reaction with BCA giving purple colour. The reading is measured at 562nm. It has the advantages of not being effected by organic solvents or detergents like guanidium chloride and urea. But it is more sensitive to reducing sugars. Another advantage it has as compared to Lowry method is that it is stable in alkali conditions so it can be done in single step.
Since it is not an end point assay, we have to make standard graph every time we take measurements after incubation for specific time at specific temperature (30 min at 37Â°C mostly).
4. Bradford method:
It is based on binding of dye Coomassie G 250 to proteins mostly arginine and lysine residues. This dye exists in four anionic forms having the pKa values as 1.15, 1.82, and 12.4 but only its anionic blue form binds to proteins absorbing at 595 nm.
It is one of the most preferred methods as it is quite fast and simple. Moreover it is sensitive and reliable and interfered by very less substances only like detergents and ampholytes. Main drawback is variation with proteins because of its binding to some amino acids only.
5. Amino acid analysis:
Most accurate method for the protein determination is the amino acid analysis. Acid hydrolysis is done and amino acid composition is determined. There is requirement for pure protein and special instrument is needed for this analysis, therefore it is not used in routine in laboratories.
6. Fluorescence assay:
Methods based on fluorescence are generally more sensitive and have more dynamic range than spectrometric methods. They may involve using of dyes which show increase in fluorescence after interacting with protein or adding some dye which binds to form a fluorescent complex with the protein.
Many such compounds are available e.g. Fluorescamine, CBQCA, OPA and nano orange.
Fluorescamine, CBQCA and OPA are non fluorescent forming fluorescent adducts. CBQCA binds to primary aliphatic amines in the presence of thiols or cyanides forming products having 450 nm excitation and 550 nm emission wavelengths. Fluorescamine complexes are measurable at 390 nm excitation and 475 nm emission wavelengths. In the presence of 2-mercaptoethanol, OPA forms fluorescent adducts detectable using 340-nm excitation and 455-nm emission wavelengths.
Tris, glycine, amine-containing buffers and the presence of free amino acids interferes with detection. These assays are very protein selective and thus show protein to protein variability.
Nano orange dye interacts with detergent coated proteins resulting in the formation of fluorescent moiety. It can detect till 10 ng/ml protein concentration. The assay is sensitive to the presence of several contaminants found such as salts and detergents, however, it is insensitive to the presence of reducing agents, nucleic acids and free amino acids. The technique is simple as the samples are heated for brief time with the dye in a detergent-containing diluent, and then cooled to room temperature. Fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths.
Other methods like Edman sequencing, HPLC, LC - MS procedures are also used for quantification of the proteins.
Many new methods and many modifications in old ones are being developed as quantitation of proteins is very important to study all biological processes and it has commercial importance in all biotech and pharmaceutical industries. When enzymes are purified, generally total amount of protein and enzymatic activity has to be monitored at each step. The proteins present in our body fluids have to be measured as urine, serum or cerebrospinal fluid for the diagnosis of the diseases.
Thus in every condition we require a highly sensitive assay giving only true signal with ease in use, less cost and not requiring use of any hazardous substances. The user is required to choose the assay for his work with utmost care and with proper knowledge of pros and cons of each assay.
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