Lab 1 Flow Chart PDF

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Flow chart required as prelab for lab 1...

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Part 1: Pepsin Digestion of Albumin

Step 1: Cut albumin (the white of a hard-boiled egg) into 16 uniform cubes of about 1-2 mm on each side.

Step 2: Place two cubes of albumin into each test tube (8 total tubes) making sure to label each with numbers 1 – 8.

1. 3 ml 5 % Pepsin solution 2. 3 ml 5 % Pepsin solution + 5 drops 1M HCl solution (DO NOT use 0.1M HCl) 3. 3 ml 5 % Pepsin solution + 5 drops 1M NaOH solution 4. 3 ml H2O (this is a control for no pepsin) 5. 3 ml 5 % Pepsin solution + 5 drops 1M HCl solution 6. 3 ml 5 % Pepsin solution + 5 drops 1M NaOH solution 7. 3 ml 5 % Pepsin solution (preboiled for 10 min) + 5 drops 1M HCl solution 8. 3 ml 5 % Pepsin solution + 5 drops 1M HCl solution + 3mL of antacid

Step 4: Gently swirl each tube to mix solutions

Step 3: Fill tubes as listed in the following box

Note: Do you think that you should be performing any other control(s) other than tube 4? Both the preboiled pepsin solution and antacid will be prepared for you to use.

Step 5: Measure the pH using pH paper. Tubes 2, 5 and 7 should be very acidic (pH = ~2) and tubes 3 and 6v should be basic.

Step 7: Measure the pH of each tube.

Step 8: Create a qualitative scale to determine the amount of protein digestion.

Step 6: Incubate test tubes 1-4 and 7-8 for 60 min at 37°C (water bath). Incubate test tubes 5 & 6 for 60 min at room temperature (about 20°C). Shake the rack gently every so often to keep evenly mixed.

Results and Analysis: 1. Place data into a table. 2. Describe the conditions necessary for digestion of albumin by pepsin. 3. Based upon your results, speculate on how you think antacids might function.

Part 2: Digestion of Starch by Amylase Starch + water

Step 1: Think about what controls you want to use for the experiment and consult with TA before setting them up.

Amylase

Maltose

Step 2: Obtain a bottle of prepared amylase (0.1% w/v) and examine the physical characteristics of the amylase (i.e. color, viscosity, pH, turbidity).

A. 2 ml amylase + 5 drops 1M HCl B. 2 ml amylase

Step 3: Label 7 test tubes from A – G and fill with the following:

C. 2 ml water D. 2 ml amylase E. 2 ml water F. 2 ml amylase G. 2 ml water

Note: Use a 5mL plastic pipette to aliquot the amylase into the test tubes and try to avoid bubbles.

Step 4: Add 2 ml of starch solution (2% w/v) to each of the test tubes for a total volume of ~4ml. Gently swirl tubes to mix.

Step 7.1: Use Lugol’s test to test for starch… Put one drop of lugol’s solution into each of tubes A1 – G1.

Step 6: After incubation divide samples into two sets of test tubes [mark A1 - G1 and A2 - G2].

A dark blue/purple colour indicates starch while shades of light grey indicate small amounts of starch.

Test one set of tubes for starch and test the other set for sugar.

Step 7.2: Use Benedict’s test to test for sugar… Add 2mL of Benedict’s solution to each tube (A2 – G2) and place tube in boiling water for 2 – 5 minutes. Refer to Benedict’s results table.

Results and Analysis: 1. Place data in table that is clear and concise and describes the results of the experiments performed. 2. Describe the conditions necessary for digestion of starch by amylase.

Part 3: Digestion and Emulsification of Lipids Emulsification: Fats/oils

---Bile-à

Tiny fat/oil droplets

Digestion: Fat/oil droplets ----Lipase-à Monoglycerides and fatty acids

Part 3.1: Observation of emulsification of fat by bile salts

Step 3: Add 2 ml distilled water to each tube.

Step 4: Add a tiny amount of bile salts to tube A only.

Step 1: Place two clean test tubes in a test tube rack and label them A and B.

Step 2: Add 1 ml vegetable oil to each tube.

Step 5: Cover each tube tightly with a square of parafilm and shake vigorously for about 1 minute.

Do you notice any difference between the two tubes? Observe for 10- 15 minutes and record your observations.

Part 3.2: Observation of lipase activity.

A. 2 ml cream and 2 ml 2% pancreatin Step 1: Place four clean test tubes on a rack and add the following:

B. 2 ml cream and 2 ml distilled water C. 2 ml cream and 2 ml 2% pancreatin and small pinch of bile salts D. 2 ml cream and 2 ml distilled water and small pinch of bile salts

Step 3: Measure and record the pH of each tubes’ contents.

Do you think the pH of the solution would affect the activity of pancreatin and why? Can it be tested under these experimental conditions?

Step 5: After incubation, test the pH and record it in table as well as note the odor of each tube.

Step 2: Cover each tube tightly with a square of parafilm and shake to mix solutions

Step 4: Incubate each tube at 37°C for up to 1 hour. Observe the changes (each 10 min.), if any, in the color of the samples. Keep track of the time of any changes....