BY PRACTICAL SKILLS IN BIOLOGY

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1 BY PRACTICAL SKILLS IN BIOLOGY Practical 3 Molecular Techniques 3 Protein Purification by Gel-Filtration and Thurs 18 and Fri 19 October 2012 CONTINUING OUR INVESTIGATION OF CHROMATOGRAPHY Some molecular biology techniques and their uses Technique Affinity Chromatography Gel-Filtration and Ion-Exchange Chromatography SDS-Polyacrylamide Gel Electrophoresis Spectrophotometric Enzyme Assay Use To purify a protein To separate molecules on the basis of mass and charge To determine the purity of a protein preparation To estimate the subunit relative molecular mass of a protein To determine the kinetic properties of an enzyme 1

2 of Oxidised and Reduced Haemoglobin Experiment 2 Separation of catalase and glucose oxidase by SAFETY PRECAUTIONS (18/19 October 2012) FERROUS SULPHATE [40mM FeSO 4 ] May cause mild eye and skin irritation. Wear gloves and take care to avoid ingestion or contact with broken skin HYDROGEN PEROXIDE [3% (v/v) H 2 O 2 ] is an aggressive oxidiser and, at high concentrations, will corrode many materials, including human skin. Wear gloves when handling this chemical. Notify the technical staff in the event of spillage. 2

3 (Gel-Permeation Chromatography / Size-Exclusion Chromatography The Theory: For animation, see next slide (A) (B) The Stationary Phase in the column consists of porous G-25 Sephadex beads. The Mobile Phase (the buffer) permeates these beads, as well as filling the interstitial space. A sample of different sized molecules is added to the column. (C) The large protein molecules, being excluded from the molecular holes in the beads, pass rapidly down the column and are eluted first. (D) The smallest molecules can move in and out of the porous beads and, therefore, move more slowly through the column. (E) Medium sized molecules penetrate the porous beads less, and move at and intermediate speed through the column. Experiment 1 (Gel-Permeation Chromatography / Size-Exclusion Chromatography (B) (C) (D) (E) 3

4 (Gel-Permeation Chromatography / Size-Exclusion Chromatography In Practice: For animation, see next slide (A) (B) (C) (D) 200µl of the FeSO 4 /Na 2 EDTA reducing agent is added to the top of the column and washed in with 500µl of phosphate buffer. As the molecules of the reducing agent are very small, they penetrate the porous beads and move very slowly through the column. Next, 500µl of oxidised sheep s blood (methaemoglobin) is added to the column and washed in with buffer. The large haemoglobin molecules are excluded from the beads and pass rapidly through the column initially through the band of the reducing agent and then into the buffer below. (E) Note what changes occur to the haemoglobin molecules as the pass through the column. Experiment 1 (Gel-Permeation Chromatography / Size-Exclusion Chromatography (A) (B) (C) (D) (E) 4

5 WHAT YOU VE GOT You are provided with the following: a tall glass chromatography column (the chromatographic bed ) assembled in a stand as shown in Figure 3.4 a glass test-tube containing a slurry of Sephadex G-25 resin in 20 sodium phosphate buffer a glass bottle containing 100 ml of sodium phosphate buffer a small glass beaker for waste a 25 ml plastic graduated cylinder a Kahn tube containing 1 ml sheep s blood, diluted 1 in 10 in buffer a brown glass bijou bottle containing crystals of potassium ferricyanide (K 3 Fe(CN) 6 ) a glass bijou bottle containing 40 mm ferrous sulphate (FeSO 4 ) a glass bijou bottle containing 80 mm sodium Ethylenediaminetetra acetic acid (Na 2 EDTA) a Kahn tube for making up the reducing agent a p200 micropipette with yellow tips a p1000 micropipette with blue tips Experiment 1 AND WHAT YOU DO (1) Pour and wash the column. (2) Oxidise the sheep s blood with crystals of potassium ferricyanide (K 3 Fe(CN) 6 ). (3) Prepare the reducing agent by mixing the ferrous sulphate (FeSO 4 ) and Na 2 EDTA. (4) Run the column, as follows: - using a p200, add 200 µl of reducing agent to the column - using a p1000, wash in with 500 µl of buffer - add 500 µl of oxidised sheep s blood - wash with buffer, and continue to add buffer until the blood has run out of the column. (5) Record your results in the Table provided. BE PREPARED THE WHOLE OPERATION MAY BE OVER IN JUST 5 MIN! (6) MOST IMPORTANT As soon as your column has finished its run, take it to one of the Technicians for cleaning IMMEDIATELY 5

6 Experiment 2 The Theory: For animation, see next slide (A) (B) The Stationary Phase in the column consists of DEAE-cellulose, a positively charged resin, in a 20mM sodium acetate/50mm acetic acid buffer (ph 5.3). A mixture of positively and negatively charged proteins are added to the column and washed in with buffer. (C) The positively charged proteins pass straight through the column, while the negatively charged proteins are bound to the cellulose. (D) A buffer of lower ph or higher negative ion (anion) concentration is applied to the column. (E) The negatively charged proteins are dislodged ( exchanged ) by the anions and are washed out of the column. Experiment 2 (B) (C) (D) (E) 6

7 Experiment 2 In Practice: For animation, see next slide (A) (B) 15ml of mould extract, containing catalase and glucose oxidase, is added to the column and washed in with 5ml of Buffer 1 (20mM sodium acetate/5mm acetic acid, ph 5.3). The negatively charged enzymes will bind to the cellulose, everything else will run through. Collect eluent in waste. (C) Add 15ml of Buffer 2 (40mM sodium acetate/40mm acetic acid, ph 4.7) to the column. Catalase will be dislodged ( exchanged ) by the buffer and washed out into the first 3 Kahn tubes (3x5ml). (D) Add 20ml of Buffer 3 (100mM sodium acetate/100mm acetic acid, ph 4.7) to the column. Glucose oxidase will be dislodged and washed out into the remaining 4 Kahn tubes (4x5ml). Experiment 2 (A) (B) (C) (D) 7

8 Experiment 2 WHAT YOU VE GOT You are provided with the following: a tall glass chromatography column (the chromatographic bed ) assembled in a stand as shown in Figure 3.5 together with 2 small balloons. a red-capped, plastic Falcon tube containing 15 ml of DEAE-cellulose resin in Buffer 1 phosphate buffer 3 plastic universal tubes containing the following: - 20 ml of Buffer 1 (20mM sodium acetate/5mm acetic acid, ph 5.3) - 15 ml of Buffer 2 (40mM sodium acetate/40mm acetic acid, ph 4.7) - 20 ml of Buffer 3 (100mM sodium acetate/100mm acetic acid, ph 4.7) a small glass beaker for waste a Kahn tube rack and a bag containing 21 Kahn tubes and a supply of Pasteur pipettes. a glass universal tube containing 15 ml of Aspergillus mould extract. FOR TESTING THE ELUENT a glass bijou bottle containing 1 g glucose, with a small spatula a light-proof, dropper bottle containing 3% (v/v) hydrogen peroxide (H 2 O 2 ) Experiment 1 AND WHAT YOU DO (1) VERY GENTLY pour and wash the column using Pasteur pipettes. (2) Run the column, as follows: - using a Pasteur pipette, add 15 ml of mould extract to the column* - wash in with 5 ml of Buffer 1. Collect eluent in the graduated cylinder. - add 15 ml of Buffer 2. Collect eluent in 3 x 5 ml Kahn tubes. - add 20 ml of Buffer 3. Collect eluent in 4 x 5 ml Kahn tubes. MOST IMPORTANT From the beginning, it will be necessary to apply pressure to force the mobile phase through the column. An inflated balloon, placed over the top of the column, will provide the equivalent of about cm of water pressure and increase the flow rate (See Figure 3.5). This will have to be removed (do not let it deflate) to allow for the addition of the buffers.. (3) Record of the colours of the ion exchange fractions collected in each of the 7 Kahn tubes, in the Table provided 8

9 AND WHAT YOU DO (4) Divide the contents of each Kahn tube equally between 3 tubes, labelled 1c, 1g, 1h, etc. (5) TESTING FOR GLUCOSE OXIDASE Add a small amount of glucose on a spatula tip to each of tubes 1g, 2g, etc. Shake to disolve the glucose, allow to stand and record your observations. Shake vigorously, and record any change. (6) TESTING FOR CATALASE Add a drop of hydrogen peroxide to each of tubes 1h, 2h, etc. Grade the catalase activity by giving 4 ticks for very active, 3 ticks for less active etc. [Note: The less active the catalase present, the longer oxygen output continues.] (7) Record all observations in the Table provided. [Note: Tubes 1c, 2c, etc. are controls.] AT THE END OF THIS PRACTICAL WASTE MANAGEMENT/ LAB CLEAN-UP (A) Today (AND ONLY TODAY) do not dispose of anything. (B) Clean and tidy-up your Workstation and Bench, leaving all of the equipment and material at your Workstation. (C) Have you Lab Manual signed by one of the Teaching Assistants. IF IN DOUBT ABOUT ANYTHING ASK FOR ADVICE 9