Lab 3 Extraction and Determination of Bacterial Proteins

Lab# 3 Extraction and Determination of Bacterial Proteins. BCH 462 [practical]

Lab#3 Extraction and Determination of bacterial protein Isolation of bacterial proteins involves several steps : 1. 2. 3. 4. 5. Growth and induction of bacterial cultures. Lysis of cells in a suitable buffer containing a detergent. DNase and RNase treatment for the removal of the nucleic acids. Determine the protein concentration using suitable method. Passage of the extract through an affinity resin and finally elution of proteins. -Biuret method, is used to determine the protein concentration, using standard curve of concentrations. - Biuret method, is based on copper ions binding to peptide bonds of protein under alkaline condition to give a violet (purple) color. -The intensity of the color resulting from the (Cu+protein) complex is linearly proportional to the concentration of protein present in the solution.

Lab #1 [Plasmid Isolation] 1) Growth of the bacterial culture. Lab #2 [Transformation of Competent Cells] 1) Growth of the bacterial culture. Lab #3 [Estimation of Protein Concentration] 1) Growth of the bacterial culture. 2) Harvesting and lysis of the 2) Harvesting. bacteria 2) Harvesting and lysis of the bacteria. 3) Purification of plasmid DNA 3)Estimation of protein concentration using Biuret method. 3)Using Ca. Cl 2 solution and brief heat shock to transform the competent cells.

B] Determination of Total Bacterial Proteins Concentration: Set up 8 test tubes as following: blank, A-E [standard BSA solution] and F-G [soluble and insoluble protein samples]. Concentration of stock solution = 140 µg/µl. Test Protein tube Distilled water Stock BSA solution Sample [µl] 140 µg/µl. [µl] concentration [µg/µl] Blank 250 - - A 200 50 - B 150 100 - C 100 150 - D 50 200 - E - 250 - F - - 250 ? G - - 250 ?