Rapid protein detection

The rapid determination methods of protein include biuret spectrophotometry, dye combination spectrophotometry, salicylic acid colorimetry, refractive index, polarimetry and near-infrared spectroscopy
.
The intelligent spectrophotometer of the new generation of microcomputer has the function of direct reading of concentration and the ability of data processing.

1.
Principle

When urea is carefully heated to 150～160℃, one ammonia molecule can be removed between two molecules to form biuret (also known as biuret).
Biuret reacts with alkali and a small amount of copper sulfate solution to produce purple red This reaction is called the double shrinkage reaction
.
Because the protein molecule contains a plate bond (—CO—NH—), which is similar to the biuret structure, it can also exhibit this reaction to form a purple-red complex.

2.
Reagents

(1) Alkaline copper sulfate solution

① The glycerol as a stabilizer: The 10mL 10mol / L KOH was added to 937mL glycerin and 3mL of distilled water, with vigorous stirring, was slowly added 50mL 40g / L of copper sulfate (of CuSO _.
4 · 5H ₂ O) solution
.

② The sodium potassium tartrate as a stabilizer: The 10mL 10mol / L KOH and 20mL 250g / L potassium sodium tartrate solution was added to 930mL of distilled water, with vigorous stirring, was slowly added 40mL 40g / L copper sulfate solution
.

When the preparation reagent is added to the copper sulfate solution, it must be stirred vigorously, otherwise copper hydroxide precipitation will be formed
.

(2) Carbon tetrachloride (CCl ₄ )
.

3.
Apparatus

(1) Spectrophotometer
.

(2) Centrifuge (4000r/min)
.

4.
Operation steps

(1) The standard curve is drawn using the sample whose protein content is measured by Kjeldahl method as the standard protein sample
.
According to the protein content of 40, 50, 60, 70, 80, 90, 100, 110 mg, weigh the well-mixed standard protein samples into 8 50 mL Nessler colorimetric tubes, and then add 1 mL of carbon tetrachloride , and then use alkali Accurately dilute 50 mL of the natural copper sulfate solution (① or ②), shake for 10 minutes, let stand for 1 hour, and centrifuge the supernatant for 5 minutes

(2) Determination of the sample accurately weigh an appropriate amount of the sample (so that the protein content is 40～110mg) in a 50mL Nessler colorimetric tube, add 1mL carbon tetrachloride, follow the above steps to develop the color, and measure its absorbance under the same conditions A
.
The measured A value can be used to check the protein quality on the standard curve, and then the protein content can be obtained from this

5.
Result calculation

In the formula, X——protein content, mg/100g

m ₁ ——The protein mass obtained from the standard curve, mg

m——sample mass, g

6.
Tips

(1) This method is less sensitive, but it is cheaper and faster than the Kjeldahl method, and can be completed in less than 30 minutes (the Kjeldahl method needs at least 2 hours to complete), and it is the simplest method for protein analysis Method
.
This Law also applies to the determination of sample pulses, oilseeds, rice and other crops seeds and meat

(2) Different types of protein have little effect on the degree of hair color

(3) After the standard curve is completed, there is no need to make the standard curve every time

(4) Samples with high fat content should be extracted and discarded with ether in advance

(5) When there are insoluble components in the sample, it will cause difficulty in colorimetric measurement.

(6) When proline is contained in the peptide chain, if a large amount of sugars coexist, the color will be poor and the measured value will be low

Related Links: Determination of Amino Acid Nitrogen in Soy Sauce