Bovine serum albumin concentration determination

Protein concentration can be determined using a method introduced by Bradford,4 which utilises Pierce reagent 23200 (Piece Chemical Company, Rockford, IL, USA) in combination with an acidic Coomassie Brilliant Blue G-250 solution to absorb at 595 nm when the reagent binds to the protein. A 20 mg/1 bovine serum albumin (Piece Chemical Company, Rockford, IL, USA) solution will be used to prepare a standard calibration curve for determination of protein concentration. The sample for analysis of SCP is initially homogenised or vibrated in a sonic system to break down the cell walls. 

Protein concentration was determined by the Bradford method (16) using bovine serum albumin as a standard. 

Miyake et al reported an ELISA method for the determination of pesticide residues in the aquatic environment. The polyclonal antibody and three monoclonal antibodies of acifluorfen were prepared by immunization of rabbits and mice with acifluorfen-bovine serum albumin conjugates. The polyclonal antibody reacted with acifluorfen at concentrations of 1.5-800 mg L , while the monoclonal antibodies reacted with acifluorfen at concentrations of 1.5-144 mg L . Among three monoclonal antibodies, AF 75-144 reacted with chlornitrofen, which did not react with the other two antibodies. It seems that the ELISA method is effective for the determination of herbicide residues in the aquatic environment. 

In this method the keyhole limpet haemoglobin conjugate was prepared as follows Keyhole limpet haemocyanin (KLH, Calbiochem, La Jolia, CA) and bovine serum albumin (BSA, BDH Chemicals) were coupled to the adduct (2), derived from 6-bromohexanoic acid and monoquat (3), via a carbodiimide reaction, as reported previously by Niewola et al. [184], The resulting conjugates contained 662mol of Paraquat per mole of KLH and 15mol of Paraquat per mole of 6-bromohexanoic acid. The amount of Paraquat bound to the protein was determined by spectrophotometric dithionite assay for Paraquat and the protein concentration was established by a standard Lowry test. 

Protein content Bradford s test was performed to determine the protein content (mg g ) of the lyophilisates. A sample of the lyophilisate (2.5 mg) was dissolved in glycylglycine (Gly-Gly) buffer (1.5 mL). An ahquot of this solution (20 gL) was diluted with glycylglycine (Gly-Gly) buffer (30 gL) and Bradford s solution (950 gL) was added. After 5 min, the absorbance was measured at 595 nm. The concentration of FSA is calculated from the interpolation of the calibration curve of bovine serum albumin (BSA) 437 mg total protein/gram of lyophihsate. 

Protein determination is necessary to estimate the amount of protein in the sample, to normalise against the protein concentration or during purification procedures. Depending on the amount of sample, accuracy and presence of interfering agents, one needs to decide on the method to be used. For accurate quantification, the sample protein is compared with a known amount of a standard protein which could either be the commonly used bovine serum albumin (BSA) or it could sometimes be immunoglobulin G (IgG). The various methods and their specifications are outlined below ... 

The working concentration of antibody mnst be determined empirically by serial dilation of the stock solution in PBSG with 10% bovine serum albumin. Usual concentrations are in the range of 10-20 pg/mL. Depending on the individual reagent, this could vary considerable. See Chapter 24 for additional instructions on performing titrations. 

The concentration of the membrane proteins is determined according to the method of Lowry et al. (39) using bovine serum albumin as the reference standard. 

Xylanase was assayed using birchwood xylan as substrate. The solution of xylan and the enzyme at appropriated dilution were incubated at 75°C for 3 min, and the reducing sugar was determined by the dinitrosali-cylic acid procedure (12) with xylose as standard. The released color development was measured spectrophotometrically at 540 nm. One unit of enzyme activity was defined as 1 pmol of reducing sugar released 1 min under the described assay conditions. Protein concentration was measured by the Lowry method (13) using bovine serum albumin as standard. 

In Bradford s method (Bradford, 1976), the OD of Coomassie Blue, a dye that absorbs at 465 nm when it is free in solution and at 595 nm when it is bound to a protein, is followed. Up to certain protein concentrations, the absorption of Coomassie Blue bound to the protein increases linearly. Determination of the protein concentration is obtained using standard proteins such as bovine serum albumin (BSA). 

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