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Several methods for determining protein content

2024-01-09 14:45

Protein is the material foundation of life, without protein, there would be no life. Protein is mainly used for maintenance, growth, pregnancy, and lactation needs. Therefore, the detection of protein content has become a routine and necessary task.

Several methods for determining protein content

1. UV spectrophotometry

Protein molecules contain conjugated double bonds of tyrosine and tryptophan, which enable proteins to have maximum absorption values for 280nm light waves. Within a certain range, the absorption value of protein solutions is directly proportional to their concentration and can be quantitatively measured. This method is simple and fast to operate, and the measured samples can be recovered. Low concentration salts do not interfere with the measurement results, so it is widely used in the biochemical preparation of proteins and enzymes. However, this method has the following drawbacks:

1. When there is a significant difference in the content of tyrosine and tryptophan residues in the protein to be tested, there may be certain errors. Therefore, this method is suitable for determining samples with similar amino acid compositions to standard proteins.

If the sample contains other substances that absorb at 280nm, such as nucleic acids and other compounds, significant interference will occur. But the absorption peak of nucleic acid is at 260nm, so the light absorption values at 280nm and 260nm are measured separately. By calculating, the interference of nucleic acid on the determination of protein concentration can be appropriately eliminated. But because the UV absorption of different proteins and nucleic acids is different, although calibrated, there are still certain errors in the measurement results.

1Bradford method

In 1976, Bradford established the principle of using Coomassie Brilliant Blue G-250 to bind to proteins, which is a rapid and accurate method for quantifying proteins. The combination of dyes and proteins causes a change in the maximum absorption light of dyes, from 465nm to 595nm. The protein dye complex has a high extinction coefficient, greatly improving the sensitivity of protein determination (minimum detection level is 1) μ g) . The binding between dyes and proteins is a rapid process that only takes about 2 minutes, and the color of the binding compound remains stable within 1 hour. Some cations (such as K+, Na+, Mg2+), (NH4) 2SO4, ethanol, etc.) do not interfere with the determination, while a large amount of detergents such as TritonX-100, SDS, etc. seriously interfere with the determination. A small amount of detergents can be eliminated by using appropriate controls. Due to its simplicity, speed, low interfering substances, and high sensitivity, the staining method has been widely used for the determination of protein content.

Determination of Total Nitrogen - Micro Kjeldahl Nitrogen Determination Method: When the measured nitrogen-containing organic matter is co digested with concentrated sulfuric acid, it decomposes into nitrogen, carbon dioxide, and water, where nitrogen and sulfuric acid are combined to form ammonium sulfate. Due to the slow progress of the decomposition reaction, copper sulfate and potassium sulfate or sodium sulfate can be added. Copper sulfate serves as a catalyst, while potassium sulfate or sodium sulfate can increase the boiling point of the digestive solution. Oxidants (hydrogen peroxide) can also accelerate the reaction.

After digestion is terminated, add strong alkaline digestion solution to the Kjeldahl nitrogen analyzer to release ammonia from the sulfuric acid ammonia component; Distill with water vapor and add ammonia to an excess standard inorganic acid solution,

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