Protein Determination Methods In general, there are the following five: Kjeldahl method, Biuret method, Folin-phenol method (Lowry method), UV absorption method and Coomassie brilliant blue method (Bradford method).
Comparison of Five Methods for Determining Proteins
method
Sensitivity
time
principle
Interfering substances
Instructions
Kjeldahl method
(Kjedahl method)
Low sensitivity, suitable for 0.2~1.0mg nitrogen with an error of ±2%
Time-consuming
8 to 10 hours
Converts protein nitrogen to ammonia and titrates with acid
Non-protein nitrogen (separable from protein precipitated with trichloroacetic acid)
Used for accurate determination of standard protein content; less interference; takes too much time
Biuret method (biuret method)
Low sensitivity
1～20mg
Medium speed
20~30 minutes
Peptide bond + basic Cu2+® purple complex
Ammonium sulfate
Tris buffer;
Certain amino acids
Used for rapid determination, but less sensitive; different proteins show similar coloration
UV absorption method
More sensitive
50～100mg
fast
5 to 10 minutes
Absorption of tyrosine and tryptophan residues in proteins at 280 nm
Various germanium and pyrimidines;
Various nucleotides
Used for the detection of column effluent; The absorption of nucleic acid can be corrected
Folin-phenol reagent method (Lowry method)
high sensitivity
~5mg
Slow speed
40 to 60
minute
Biuret reaction; Phosphomolybdic acid-phosphotungstic acid reagent is reduced by Tyr and Phe
Ammonium sulfate
Tris buffer;
Glycine
Various mercaptans
It takes a long time; operation must be strictly timed;
Color depth varies with different proteins
Coomassie brilliant blue (Bradford method)
Highest sensitivity
1～5mg
fast
5 to 15 minutes
When coomassie brilliant blue dye binds to protein, its lmax changes from 465nm to 595nm
Strong alkaline buffer;
TritonX-100;
SDS
The best way;
Less interfering substances
Color stability
Color depth varies with different proteins
From the above table, it can be concluded that different methods have different characteristics and advantages, such as the UV absorption method to determine the time quickly. But in the end, the last kind of Bradford method has the highest efficiency, and it is comparatively dominant whether it is time, sensitivity, or interference.
Below, we will focus on one of the protein determination methods - Kjeldahl method.
Kjeldahl method (Kjedahl method)
First, pour concentrated sulfuric acid into the sample and shake it side by side so that the concentrated sulphuric acid is in full contact with the sample. Then heat the reagent. If you need to speed up the experiment, you can add CuSO4 as a catalyst, so that the experimental reaction time will be greatly reduced. CH2COOH reacts with H2SO4 to produce ammonia, which then reacts with H2SO4 to fix nitrogen. At this time we got the solution of (NH4)2SO4, then we can use NaOH to react with ammonium sulfate. By calculating the amount of NaOH used, we can calculate the nitrogen content. The following is the chemical reaction that occurs in the entire nitrogen determination process.
CH2COOH+ 3H2SO4 = 2CO2 + 3SO2 +4H2O + NH3 (1)
2NH3 + H2SO4= (NH4)2SO4 (2)
(NH4)2SO4 + 2NaOH =2H2O +Na2SO4 + 2NH3 (3)
When we get the nitrogen content, we can finally calculate the protein content through a certain calculation.
The Kjeldahl method is commonly used for the determination of nitrogen content in organic compounds. It is a classical method for the determination of protein content. Although the amount of reagents used during the test is large, it is undeniable that the wide range of samples it can be applied to is tested. The accuracy of the results is recognized.
Now, with the development of science and technology, we have developed the Kjeldahl nitrogen analyzer with a digester, which completes the first two steps of the nitrogen determination process through the instrument, facilitating the implementation of the entire process. The Kjeldahl analyzer automatically digests the sample, distills it, and intelligently completes it, saving time and effort and improving work efficiency. This protein determination method has the advantages of accuracy, rapidity, simplicity, low consumption, and stability. Zhejiang Top Instrument Co., Ltd. provides this product, 24-hour service hotline.
Kjeldahl
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