explained
Soluble Protein means a state of a small molecule may be water-soluble proteins or other solvents. Often used as an important indicator in plant physiology, microbiology, food processing and other experiments. The soluble protein is one of the important indicators of drought resistance of plants.
test measurement-250 G Method Brilliant Blue principles
Coomassie Brilliant Blue G-250 (GooMAssIe BrIllIAnT Blue G-250) Determination of protein content belonging dye-bindingof law. Coomassie Brilliant Blue G-250 in the free state was red, in dilute acid solution, when it is combined with the hydrophobic region of a protein becomes blue, the former maximum absorption of 465 nm light, the latter 595nM. Protein concentrations within a certain range (1 ~ 100μg), pigment-binding protein was proportional to the light absorption at the wavelength of 595nM and protein content, it can be used for quantitative determination of proteins. Protein with Coomassie Brilliant Blue G-250 binding in about the 2Min equilibrium, the reaction is completed very rapidly, stable over 1H combinations thereof at room temperature. The reaction is rapid and sensitive (sensitivity further four times higher than FolIn- phenol method), easy to operate, less interference substance (Coomassie brilliant blue G-250 and protein bound by van der Waals forces, less influenced by the specific protein, in addition to histones outer , various other types little protein staining intensity difference), can be determined microgram protein content, the protein is a better quantitative method. However, this method also has its disadvantages, Coomassie brilliant blue at high linear low protein content, and different sources of protein and pigment-binding conditions are different.
instruments and appliances
721 spectrophotometer; 10 Ml cylinder 1; mortar; beaker; flask; pipette:? 1Ml 3 branched, 0 1Ml 3 branched; 10 Ml stoppered graduated test tube 14.
reagent
standard protein solution: Bovine serum albumin formulated with 100μg / Ml standard protein solution containing the protein;
90% ethanol;
85% phosphoric acid (W / V);
Coomassie brilliant blue G-250 solution: Weigh 100μg Coomassie brilliant blue G-250, was dissolved in 50Ml 90% ethanol, 85% 100 mL phosphoric acid, and finally with distilled water to 1000ml, storage is in a brown bottle, the solution may be at room temperature for 1 month.
Operation
1? Standard curve (protein content of 0 ~ 100μg / Ml) 6 taken stoppered test tube, prepared according to Table 2-1 data containing 0 ~ 100μg / Ml bovine serum protein solution each 1Ml.
Table 2-1 different protein content of bovine serum protein was formulated in Table
123456 pipe protein standard solution (ml) 00.200.400.600.801.00 distilled water (ml) 1.000.800.600. 400.200 protein content (μg) 020406080100 5Ml after adding Coomassie brilliant blue G-250 solution, stoppered, inverted several times to mix, placed in 2min, with a light path cuvette 1CM color at 595nM in each tube. A protein concentration of abscissa and the ordinate is absorbance standard curve.
2. Determination of protein concentration in the sample extract draw 1.0 mL sample extract (see LoWry sample extraction method), placed in stoppered test tube (in duplicate for each sample set), added 5Ml test brilliant blue G-250 solution, mixed well, after placing 2Min color at 595 nm, recording absorbance and the standard curve obtained by the protein content.
3. Results calculated
in the protein content of the sample (Mg / g) = (C × VT) / (V1 × FW × 1000) (2-2)
where C: check the standard curve values (μg); VT: extracting the total liquid volume (Ml);
FW: sample fresh weight (g); V1: amount of sample measured ( ml).