BIOCHEMISTRY

Biochemistry of macromolecules & metabolic pathways work book

DateTask 1

Definition

The Bradford protein assay is one of several simple methods commonly used to determine the total protein concentration of a sample. The method is based on the proportional binding of the dye Coomassie to proteins. Within the linear range of the assay (~5-25 mcg/mL), the more protein present, the more Coomassie binds. Furthermore, the assay is colorimetric; as the protein concentration increases, the color of the test sample becomes darker. Coomassie absorbs at 595 nm. The protein concentration of a test sample is determined by comparison to that of a series of protein standards known to reproducibly exhibit a linear absorbance profile in this assay. Although different protein standards can be used, we have chosen the most widely used protein as a standard - Bovine Serum Albumin (BSA) (Miron, 2000).

Features1 Fast: 10 minutes can complete the determination. 2 Stable: pipette, mix 2 minutes either measured absorbance change within 1 hour of not more than 10%. 3 High Sensitivity: can be precisely quantified 1 ~ 1000 µg / ml of protein sample. 4. 595 nm detection 4 samples from most of the effects of chemical substances. Mercaptoethanol concentrations are up to 1M, dithiothreitol concentration up to 5 mM. If SDS than 0.1%, TritonX-100 more than 0.1%, 20, 60,80 than 0.06%, preferably small sample was diluted to an appropriate concentration with water, re-determination.

1: Coomassie Blue ReagentCoomassie Brilliant Blue G-250 100mg was dissolved in 50ml 95% ethanol, 100ml 85% H3PO4, diluted with distilled water to 1000ml, filtered through filter paper. Final reagent containing 0.01% (W / V) Coomassie Brilliant Blue G-250, 4.7% (W / V) ethanol, 8.5% (W / V) H3PO4. 

2: Standard protein solution

Pure bovine serum albumin, according to its purity with 0.15mol / L NaCl preparation of 100 ug / ml protein solution. 

Another two clean tube (make a duplicate), adding the appropriate concentration of test sample, the measured value to the range of the standard curve, measured as above, the absorbance of the sample solution from the standard curve can be calculated to check the content. (Itzhaki, 1964)

Purpose of the experiment

Preparation of a concentration of 0.10mg/ml, 0.08mg/ml, 0.06mg/ml, 0.04mg/ml, 0.02mg/ml, 0 mg / mlbovine serum albumin (BSA) was measured absorbance of the solution in this group to give a protein concentration standard curve of absorbance. Unknown protein concentration was measured absorbance of the sample, the standard curve to obtain the concentration of protein (Bradford, 1976).

Experimental principle

     Coomassie Brilliant Blue (CBB) determination of protein content is a dye-binding assay. Coomassie Brilliant Blue in the free state, red, maximum light absorption at 488nm; when it is combined into a cyan protein, protein - dye conjugate at 595nm wavelength of maximum absorption. The light absorption is proportional to the protein content, it can be used for the quantitative determination of protein. Binding protein with Coomassie Brilliant Blue in 2min or so to reach equilibrium, the completion of the reaction is very rapid; their conjugates 1h at room temperature remains stable. The Act reagent preparation is simple, easy operation, the reaction is very sensitive, the sensitivity is higher than the Lowry method four times, can be determined microgram protein content determination of protein concentration range of 0 ~ 1 000µg/ ml, is a commonly used trace ...