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Kaomas bright blue G-250 methodprotein
(70,135,221); "> "principle"
Kaomas bright blue method to determine protein concentration, It is a fast and sensitive method of quantitative determination of trace protein concentration by using the principle of protein-dye binding.
there are two different color forms, red and blue, in the Coomas Bright Blue G-250. The Kaomas Bright Blue G-250 is brownish-red in acidic free state, with a maximum light absorption of 465nm, when it binds to proteins, and a maximum light absorption of 595nm.
in a certain protein concentration range, the protein-dye complex at a wavelength of 595nm light absorption is directly related to the protein content, by measuring the increase in light absorption at 595nm can be known with the amount of protein binding to it.
protein and coomas bright blue G-250 combined to achieve equilibrium in about 2min time, complete the reaction very quickly, its binding at room temperature for 1 hour stable. The protein-dye complex has a high de-lighting coefficient, which makes it highly sensitive to determine protein concentration and can measure microgram protein content.
"methods and steps"
1, standard curve drawing
take 6"", press the table to add each .
tube number " | 0 | 1 | . 2 | . 3 | |
5
100 sg/ml cow serum albumin solution/mL
0.2
0.6
0.8
1.0
distilled water/mL
1.0
0.8
.
0.4
;">0.2
Kaomas bright blue liquid/mL
5.0
5.0
5.0
5.0
5.0
5.0
after adding the Coomas bright blue G-250 protein reagent, shake well, place 2min, at 595nm wavelength color measurement, record A595.
draws the standard curve with the corresponding standard protein content (mg) as the horizontal coordinates and A595 as the ordinates.
2, sample determination
test tube Add homemade protein sample 1.0mL, then add 5.0mL Kaomas bright blue G-250 reagent, shake well, place 5min, at 595nm wavelength color, record A595.
< p style is "text-align:left;" > protein content is found on the standard curve based on the A595 measured.notes
(1) If the measurement requirements are strict, light absorption can be measured within 5 to 20min after the reagent is added, because the color is the most stable during this time. The color reaction needs to be completed within 1h.
(2) assay, the protein-dye complex will have a small amount of adsorption on the wall of the color cup, and experiments have shown that the adsorption of this compound can be ignored. After the determination, the blue color cup can be washed clean with ethanol.
.