Calculation method for acridine ester (NSP-SA-NHS) labeled protein

 

Acridine ester (NSP-SA-NHS) labeled protein is one of the commonly used experimental methods in biological and biochemical research, which can bind proteins to fluorescent dyes, facilitating detection and quantitative analysis in experiments. When conducting acridine ester labeled protein experiments, we need to calculate parameters such as acridine ester concentration, protein mass concentration, and F/P molar ratio to ensure the accuracy and reliability of the experimental results. The following will provide a detailed introduction to the calculation method of acridine ester labeled proteins.

 

1. Prepare the sample to be tested: Take 100 μ Dilute the sample to Abs280 between 0.1 and 1.5 as needed. This step is to ensure that the absorbance values obtained in subsequent experiments are within the appropriate range to ensure the accuracy of the calculation results.

2. Adjust pH value and measure absorption peak: Add a small amount of hydrochloric acid to the diluted sample, adjust its pH value to the range of 1-2, and form a salt solution with yellow characteristics. Then measure the absorption peak position of the solution, usually located at 367nm.

 

3. Measurement of absorbance value: Measure the absorbance values of the sample to be tested separately, including Abs280nm and Abs367nm.

 

4. Calculate the concentration of acridine ester: Based on the obtained absorbance value, use the following formula to calculate the concentration of acridine ester: acridine ester concentration (mol/L)=Abs367/14650

 

5. Correction of protein absorbance value: Use the following formula to correct the absorbance value of protein: Abs280 (after correction)=Abs280- (Abs367 ×  0.17)

 

6. Preparation of unlabeled protein samples: Prepare 1mg/mL unlabeled protein solution, then take 10 µ L, add 50 µ L labeled buffer, and dilute with 490 µ L deionized water. Read the absorbance value of the diluted solution at Abs280nm and multiply it by the dilution factor.

 

7. Calculate protein mass concentration: Based on the diluted absorbance value, use the following formula to calculate protein mass concentration: protein mass concentration (mg/mL)=absorbance value (corrected) ×  Dilution ratio/molar absorption coefficient of protein

 

8. Calculate the F/P molar ratio: Use the following formula to calculate the F/P molar ratio: F/P molar ratio=acridine ester concentration/protein molar concentration

 

Through the above steps, we can accurately calculate various parameters of acridine ester labeled proteins, including acridine ester concentration, protein mass concentration, and F/P molar ratio. These calculation results will help us understand the efficiency of protein labeling and obtain accurate quantitative analysis results in experiments. During the experimental process, it is necessary to strictly follow the experimental steps and pay attention to avoiding errors in the operation to ensure the reliability and accuracy of the experiment. As an advantageous manufacturer of luminescent reagents, Desheng can provide six different groups of acridine ester raw materials, which are simple to prepare, fast to use, and can be effectively applied in various experiments. If you are interested, please feel free to contact us for purchase!