标准蛋白曲线制作（Standard protein curve）.docVIP

标准蛋白曲线制作（Standard protein curve） The standard curve made by Coomassie brilliant blue method measurement of protein content Standard curve Generally, the content of the substance is measured by spectrophotometry. The standard curve is first made, and then the content of the substance is determined according to the standard curve. Therefore, the production of standard curves is a basic technique in biological detection and analysis. Two. Determination of protein content 1, Kjeldahl method 2, biuret method 3, Folin- phenol reagent method 4 、 UV absorption method 5, the method of Coomassie brilliant blue Determination of protein content of three - standard production curve, the method of Coomassie brilliant blue (I) reagents: 1, Coomassie blue reagent: Kaumas blue G - 250 100mg 50ml dissolved in 95% ethanol, adding 100ml 85% H3PO4, diluted with distilled water to 1000ml filter. The final reagent containing 0.01% (W/V) G - 250,4.7% Kaumas blue (W/V) ethanol, 8.5% (W/V) H3PO4. 2 standard protein solution: Pure bovine serum protein, protein nitrogen content determination in advance by the micro Kjeldahl method, according to its purity with 0.15mol/LNaCl preparation of 100ug/ml protein solution. (two) equipment: 1 and 722S spectrophotometer. 2 、 pipette. (three) standard curve making: Test tube number 0123456 100ug/ml standard proteins (ML) 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 0.15mol/L, NaCl (ML) 1, 0.9, 0.8, 0.7, 0.6, 0.5,, 0.4 Coomassie blue reagent (ML) 5 5 5 5 5 5 5 Shake well. Within 1h, take No. 0 tube as blank control and color at 595nm 1, A595nm 2, with A595nm as ordinate, standard protein content as abscissa (six points for 10ug, 20 UG, 30 UG, 40 UG, 50 UG and 60 UG), plotted on the axis of the standard curve. 1) use standard curve to find regression equation. 2) calculate regression equation by formula. 3) or draw the linear regression equation by origin mapping. That is, A595nm=a * X () +6, the general correlation coefficient should be more than 0.999, at least 2 mor