Protein concentration determination - double reduction.

urea heating, Two molecules of urea release a molecule of ammonia to form a double peptide (NH2-CO-NH-CO-NH2).

double-shrink in an alkaline solution in combination with Cu2 plus to create a purple enses, a chromatin reaction called a double-shrink reaction. The peptide bonds in protein molecules are similar to the structure of double-shrink, so they can also react with double-shrink.

in a certain concentration range, the color depth is linearly related to protein content. The higher the protein concentration, the darker the color of the system. The reaction product has maximum light absorption at 540nm.

reacts with a sample solution of unknown concentrations and a series of known concentrations of standard protein solutions simultaneously with a series of known concentrations reagents and, at 540nm color, the concentration of unknown sample proteins can be obtained by the standard curve drawn by the standard concentration protein.

all contain a peptide bond and a -CSNH2, -CRHNH2, -CHNH2-CHNH2-CH2OH, -CHOH-CH2OH, -CHOH-CH2NH and acetyldiamines.

method is simple to operate, fast, protein concentration and light density linear relationship is good, is one of the common methods of protein concentration analysis, but the disadvantage of this method is low sensitivity, protein quantity must be 1 to 20 mg / mL side has better results. This method is commonly used in assays that require fast but low accuracy.

"methods and steps"

standard curve drawing

< "text-align: left;" > take 6test tubes from 0 to 5 numbers, according to the table to add each reagent, each add a test fluid must be mixed, after addition at room temperature for 30 minutes, and then 0 tube as a blank control tube, 540nm to determine the tube A value.

1

3

4

5

standard casein/mL

distilled water/mL

double-shrink reagent/mL

0

<:left;">1.0

4.0

0.4

0.6

4.0

0.4

4.0

0.8

0.2

4.0

1.0"

0

4.0

with standard casein (mg) as the horizontal coordinates, A 540 as the ordinate, draw the standard curve.

unknown concentration of sample solution determination

can be extracted from milk casein preparation solution. Sample determination of unknown casein concentration 3 tubes. The method step is the same as that of tube 5 of the above standard curve production process, replacing the standard casein with only 1.0mL of the unknown concentration casein solution.

, based on the A540 value of the unknown concentration casein test fluid, found the corresponding number of protein milligrams on the standard curve, i.e. the mass concentration (mg/mL) of the casein solution under test.