Protein quantification of any sample is all about determining the protein content. It helps in allowing you to load the amount of protein in every lane. This also helps in preventing overloading the lane. There are different ways that can be used to quantify the concentration of protein in samples. One of the best methods of quantifying is spectrophotometric determination. This method of measuring protein content is considered highly popular. In this method of quantification, there’s no use of hazardous agents which makes it high in demand.
Along with spectrophotometric determination, there are many other methods through which quantification of protein is performed in labs. Selection of the quantification method is completely dependent on different factors like cost-effectiveness, reproducibility, and ease of use. Most importantly, all the methods should quantify the protein concentrated samples in a very precise way. The sensitivity level of the analysis process helps in determining a protein’s lowest level. Different assays of protein quantification have different specificities. Now, let’s discuss them along with their advantages.
Biuret way of quantification
This sort of test is mainly performed for checking out how much peptide bonds are present in the sample. In this test, highly stable Cu2+ tends to create a violet-colored complex. This usually happens under alkaline conditions. Throughout the process, the concentration of protein gets absorbed at 540 nm as it is proportional to the color intensity. This method for quantification is generally preferred for those concentrations where the high protein is found.
The dye-binding way of quantification
Under this assay, different dyes can be used for performing quantification process such as bromocresol green and eosin. Nowadays, Bradford assay is considered the best and commonly used dye. The reason for choosing this method is its excellent sensitivity and accuracy that gives a precise result for protein quantification. It has compatibility with various buffers. Also, it can be performed in a quick way.
The Lowry way of quantification
The main advantage of using this method is that it is cost effective and has the ability to be reproduced. In this method, the sample is heated with a reagent that is Folin-Ciocalteu. If the conditions are alkaline, this reagent gets reduced which results in vigorous blue color. This method is best suited for the concentrations that range from 0.01 mg/mL to 1.0 mg/mL.
Bicinchoninic Acid way of quantification
This method of assay is somewhat similar to the method in which reagents are used. In this assay, bicinchoninic acid is used as a reagent for the quantification process. The whole assay process is performed at extreme temperatures for reducing the variability and maximizing the sensitivity. Like Lowry quantification process, this method of analysis has also the ability to be reproduced.
Quantification of protein is extremely necessary as it helps in calculating the total protein content in a sample. In simpler words, it can be said that the main purpose of the protein assay is to find out protein amount in any concentrated sample. For this, there are various methods of quantification like dye binding methods. Visit www.biochain.com/products/protein/total-protein-lysate/ for more information on total protein tissue lysates.