A comparison of methods for determining total body protein

Area of Science:

• Biochemistry
• Animal Physiology
• Analytical Chemistry

Background:

• Accurate quantification of total protein is crucial for understanding physiological states in animal models.
• Standard protein determination methods may vary in efficiency and accuracy depending on the biological matrix.

Purpose of the Study:

• To identify the most convenient and accurate method for determining total protein in whole-body rat homogenates.
• To compare different protein extraction and quantification techniques against a reference standard.

Main Methods:

• Evaluated three protein extraction methods: water, 6 N guanidine-HCl, and 5% (w/v) sodium dodecylsulfate (SDS) in 0.5 N NaOH.
• Assessed five protein concentration methods: Biuret, Bradford, bicinchoninic acid, and Kjeldahl.
• Compared results against total amino acid analysis following acid hydrolysis as the reference value.

Main Results:

• Extraction with 5% SDS in 0.5 N NaOH proved superior to water or guanidine-HCl.
• The Biuret method, applied to SDS-NaOH extracted samples, yielded results 90% of the reference value with low variation (2.4%).
• The Kjeldahl method required a specific nitrogen factor (5.51 ± 0.03) for accurate protein concentration determination.

Conclusions:

• Extraction using SDS-NaOH followed by the Biuret assay is a highly effective and recommended method for quantifying total protein in rat whole-body homogenates.
• This combined approach offers a balance of accuracy and practicality for researchers.
• Optimization of extraction and quantification is key for reliable biochemical analysis in animal studies.