Protein binding assay with cyanogen bromide-activated agarose
Protein binding assay with cyanogen bromide-activated agarose
This is an effective method for covalently immobilizing enzymes or proteins, such as peroxidase or antibodies, in agarose or cross-linked dextran. A serious disadvantage of this method is the high toxicity of the reagents used, and it is recommended to use products purchased from companies (e.g. CNBr-activated agarose). Since activation needs to be done under strong alkaline conditions, the structure of the gel is likely to be damaged, so it should be done quickly. Source: Laboratory Manual of Enzymology
Operation method
basic program
Materials and Instruments
Protein Move See "Other" for "Reagents" required for the experiment. 1. Preparation of CNBr-activated agarose 4B The following steps must be performed carefully in a fume hood. Adjust the agarose 4B suspension to pH 11.2 with 3 mol/L NaOH and check with a pH meter. Place a flask on a balance, zero the weight, dissolve 0.67 g of solid CNBr in a small amount of water and carefully stopper the flask. Carefully add the CNBr solution to the gel suspension while stirring (KPG or spiral stirrer, do not use a magnetic stirrer), keeping the pH at 11.2, which can be adjusted with 3 mol/L NaOH. When the pH has remained constant for 6 min, immediately rinse the gel with 1 L of water through a Brinell's funnel or glass. 2. Conjugated proteins Rinse the CNBr-activated agarose suspension (20 ml) and transfer to the same volume of 0.1 mol/L NaHCO3 solution, pH 8.2. Add 0.5 mol/L NaCl and 0.2 g protein. The suspension was shaken for 3 h at room temperature or overnight at 4°C. The protein content of the supernatant was determined. The amount of protein immobilized is determined by measuring the protein content of the supernatant. Add 5 ml of 1 mol/L aminoacetic acid to sequester the remaining reactive groups. The gel is first rinsed with 0.1 mol/L pH 4.0 sodium acetate-acetic acid and then with 0.1 mol/L pH 7.6 potassium dihydrogen phosphate. To store the gel, NaN3 was added at the end to reach a final concentration of 0.1 mol/L. Common Problems Reagents: Agarose 4B, suspension, 6.6 g dissolved in 20 ml H2O 3 mol/L NaOH (Mr = 40.0; 12 g dissolved in 100 ml water) Cyanogen bromide (CNBr, Mr = 105.9) 0.1 mol/L NaHCO3 (Mr = 84.0; 0.84 g/L 100 ml) pH 8.2 with 0.5 mol/L NaCl (Mr = 58.4; 2.92 g/100 ml) 1 mol/L aminoacetic acid (Mr = 75.1; 1.5 soluble in 20 ml H2O ) 0.1 mol/L sodium acetate-acetic acid, pH 4.0 0.1 mol/L potassium dihydrogen phosphate, pH 7.6 For more product details, please visit Aladdin Scientific website.
Agarose 4B NaOH Cyanogen bromide NaHCO3 Aminoacetic acid Sodium acetate-acetic acid Potassium dihydrogen phosphate
Balance Flask Spiral stirrer Brinell's funnel