Assessing starch digestion by salivary amylase lab report

TOPIC 2: ENZYME DATA

BIO 330L General Physiology 2015 Page 1

TOPIC 2 Enzymes & Protein Digestion Enzymatic Digestion Chemical digestion in mammals begins in the mouth. Saliva from the salivary glands contains mucus for lubrication, water to dissolve the food so that we can taste it, and salivary amylase (ptyalin) for carbohydrate digestion. Amylase breaks down starch from plants into maltose, a disaccharide, and into small polymers of glucose. Another plant glucose polymer, cellulose, is not digested by humans and remains in the digestive tract as fiber or roughage. The pH of the mouth is nearly neutral (about 6.7). Food leaving the mouth and now combined with amylase, reaches the stomach where the pH changes dramatically due to secretion of hydrochloric acid from parietal cells in the wall of the stomach. The pH of the stomach is usually in the range of 1-4. Amylase is inactivated at low pH, while the inactive protease pepsinogen from the chief cells of the gastric mucosa is converted to its active form, pepsin. Pepsin breaks peptide bonds of proteins to form smaller polypeptides. From the stomach, the food-enzyme mixture (chyme) enters the small intestine. It is here that most digestion and absorption takes place. The highly acidic chyme is neutralized (raised to pH 7) by secretion of bicarbonate from the pancreas; the change to this pH also inactivates pepsin. Digestion continues with the secretion of additional enzymes from the pancreas and from isolated endocrine cells in the wall of the small intestine. Lactase, maltase, peptidases, and nucleases are all enzymes secreted by the intestine. The pancreas secretes more amylase for carbohydrate digestion, proteases such as trypsin, and lipase for fat digestion. The monosaccharides (e.g. glucose) and amino acids from carbohydrate and protein digestion, go directly into the hepatic portal vein, and from there to the liver to be used in catabolic or anabolic metabolism. Assessing Enzyme Activity Any given enzyme has some conditions that yield optimum activity. To test the hypothesis that a particular enzyme has a particular pH or temperature optimum, you can determine the activity of the digestive enzyme at several temperatures and under acidic, neutral and basic conditions. A common way to assess an enzyme's activity is to combine appropriate substrate (i.e., starch, protein, fat) with an enzyme that digests this substrate. Then you calculate if and/or how much of certain products are produced (and/or how much of the substrate remains, depending on the availability and ease of these tests). One way to assess amount of either a substrate or a product molecule is to use a reagent that is converted chemically to a colored derivative in the presence of a specific compound (e.g., the substrate; or the product). Because these cause color changes that indicate the presence of specific molecules, these are called "indicator solutions". Using spectrophotometry, you can then quantitatively measure the amount of the colored derivative produced by the reaction with the indicator solution. You will assess activity of the enzyme pepsin, which breaks peptide bonds of proteins to form smaller polypeptides. In testing the activity of pepsin you will use the Bradford-Coomassie reagent. In the Bradford test, compounds react with any peptide chain having with a minimum of three amino acids and more color indicates the presence of a peptide bond between two CONH, groups in a protein or peptide. This is a quantitative indicator of the number of peptide bonds still present.

A. TYPES OF PIPETTES

1. Glass or plastic pipettes: a. Note total delivery volume or range possible (20ul, 200ul, 1 ml, 2 ml, 5 ml or 10 ml, etc.). b. On pipettes: Note how many divisions there are AND what they represent. c. Some pipettes are numbered with 1 starting at the top, others start with 1 at the bottom (tip). d. Some will indicate “TD” versus “TDX”, printed (at the top):

2. TDX Pipettes. TDX means “to deliver with exhale” (from the days mouth-pipetting was in practice). This means the pipette will deliver the calibrated volume whe ALL the volume is expelled (e.g. pushed out w the pipet bulb). READ section below for “TD” pipets.

3. “To Deliver” (TD) Pipettes a. When amount has been delivered, touch the inside of the vessel with the tip of the pipette to knock

off the drop on the tip.

II. TECHNIQUES

I. GENERAL BACKGROUND

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BIO 330L General Physiology 2015 Page 2

o Teams A, C, E are assigned the pH hypothesis. o Teams B, D, F, G are assigned the Temperature hypothesis

b. The small amount remaining inside the tip and adhering to the sides has been accounted for in the calibration and should remain inside.

4. Micropipettors: for < 1 mL (ie up to 1000 uL) a. Some are fixed volume (e.g. 100 uL = 0.100 ml). Others are adjustable, so be careful to double-

check the volume setting. Good working habit at to always check the setting each time you pick up the pipette and ALSO when just about to use it.

b. These pipettors have disposable plastic tips. Tip must be placed on firmly to pipette correct volume. Tips are ejected with a moveable collar that is depressed down onto the rim of the tip by a lever.

c. Pre-wet tip by slowly taking up solution. (Ultra-micropipettors- usually below 15 uL, usually are NOT calibrated with pre-wet tips-CHECK the INSTRUCTION BOOK if you don’t know) and then slowly ejecting the solution.

d. How much you control the down- and upward movement of the plunger greatly affects accuracy. e. With a prewet tip, immerse tip just under surface of fluid (about 1-2 mm) and “aspirate (take up) the

desired volume, controlling the upward movement of the plunger with your thumb. f. Dispense solution slowly (again, controlling the downward movement) and evenly against side of the

tube (clean area). g. PRACTICE THIS SEVERAL TIMES. If you get air bubbles in the tip you are letting the plunger move

too quickly.

B. OTHER LAB PROCEDURES

 Before using anything, LABEL ALL Glass PIPETTES WITH NAME OF SOLUTION. Also label an

unlabel flask etc. READ the labels on the flasks you get from the front table.

 Contamination. Think carefully about what you are doing. It is very easy to create contamination, either of STOCK solutions, or of solutions you have on your work bench. If in doubt, get another pipette tip or disposable (glass) pipette.

 STOCK pH BOTTLES in front of room. Contamination is a real concern for the enzyme lab. For each pH buffer, use the dedicated PIPETTS to dispense a little more than the total vol you will need into, a labeled Test-tube or set of test tubes or flasks (clean flasks are on a tray at table). Do the pipetting into digest test tubes at your lab bench.

 R/O water is in the D/I label taps. At each table. Fill up and use your glass flask (labeled either “R/O” or or “DW”) for any use requiring pipetting with R/O water.

 DO NOT use the vortexer with wet test tubes (electrical hazard); wipe tubes dry first.

 WASH your GLASSWARE except test-tubes (instructions at end of handout). Return to the tray on your table to dry.

III. COLORIMETRIC ASSESSMENT OF ENZYME ACTIVITY: TESTING HYPOTHESES ABOUT ENZYME ACTIVITY

Experimental and Control tubes: An experimental tube is one where you manipulate the variable that (in your hypothesis) you think has an effect on the outcome. In the enzyme study, you should consider how to demonstrate that a digestion reaction will not take place unless a specific content and/or a certain condition is present. Thus, for an experimental tube, certain contents or conditions are omitted from the test tube prior to incubation. Each experiment should also have a number of vials that are controls for the experiment. Controls can have a variety of specific purposes; in general the function of a control is to show that the expected outcome occurs because of the variable you manipulated, and not some other effect. In addition, controls are also used to indicate contamination: if you get reaction products in your tube when you should not have, it indicates contamination. (team IDs are the TABLE Letter on the front (North) face of the table)

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Temperature Teams: What would be a specific statement (hypothesis) about the effects of temperature on the actions of the proteolytic enzyme, pepsin? You should first read about this enzyme in your text book. The optimal pH for the reaction is what you will use and so it is not a variable. We can provide the following three temperature conditions: room temp (23 ºC; warm (30 ºC) and hot (37 ºC ) water baths. You are also testing that the enzyme itself is involved in the digestion (not just that there is an optimal temperature for this enzyme). You will need to state this hypothesis in your Report. pH Teams: What would be a specific statement (hypothesis) about the effects of pH on the actions of the proteolytic enzyme, pepsin, based on your reading about this enzyme? Assume you know the optimal temperature for the reaction and it is not a variable. The pH of the reaction tube can altered by adding pH buffers. We have buffers for: pH 4 through pH 9. To definitively test your hypothesis, you will need to set up several types of reactions (test-tubes) that differ in their content/conditions. We have enough reagents to allow you to test THREE different pH values. Think of what control treatments are needed: You are also testing the simple hypothesis the enzyme itself is involved in the digestion (not just that there is an optimal pH for this enzyme).

BACKGROUND INFORMATION: The protein substrate we will use is BSA (bovine serum albumin), commonly used in many laboratory procedures. The BSA (protein substrate) solution a 20 mg/ml solution. Enzyme Pepsin catalyzes the breaking of peptide bonds of proteins to form smaller polypeptides. The enzyme is a 5% solution (a “X percent solution is defined as “X g per 100 ml solute”). BioRad Dye Reagent

 This reagent is based on the Bradford protein assay, which uses the Coomassie dye.

 This dye (Brilliant Blue G250 in an acidic solution) binds primarily to basic and aromatic amino acid residues.

 When bound, the absorbance maximum shifts from 465 to 595 nm.

 The Bradfood Coomassie reaction is sensitive for protein concentrations from 1 to 10 mg/ml; it is linear for BSA from 0.2 mg/ml to 0.9 mg/ml. It is a little curvilinear beyond.

 The Bradford Protein assay is fast, but it is sensitive to affects of non protein sources, such as detergents. The response (absorbance) varies with the type of protein; it is very sensitive to the substrate we are using, the Bovine Serum Albumin (BSA) protein.

Procedure Overview (see Set-up & Data Table on pages 9 & 10)

 Part A: Digestions: Mix the Protein Substrate (20 mg/ml bovine serum albumin, BSA) with an enzyme (5% pepsin) that digests proteins, a pH buffer (or an isotonic diluent), and incubate in a water bath for a set time to allow digestion.

 Part B: Standard Curve: Determine absorbance (OD) of known concentration of the BSA solutions (standard curve tubes, each in triplicate).

 BRADFORD TEST: For all tubes in Parts A or B, you cool to room temp, run through the Bradford Test and measure the OD of each. You don’t have to do them all together.

 In Excel, enter the data for known concentrations and make a graph to produce your Standard Curve. This curve is hyperbolic, but over this lower concentration range is almost a straight line.

 Use a printout of the standard curve to determine amount of protein still in samples.

 The reagent produces a reaction that is a quantitative indicator of the number of peptide bonds present; the more blue the solution, the less digestion has taken place (there is still a lot of protein present). Recall that when a protein is digested, amino acids are produced, which make the solution acidic.

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TOPIC 2: ENZYME DATA

BIO 330L General Physiology 2015 Page 4

ENZYME DIGESTION EXPERIMENT tube

(Part A)

Set up reaction tubes (in triplicate):

20 mg/ml BSA Substrate

+ pH Buffer + Enzyme OR diluent ((0.9%

NaCl)

Vortex

Incubate 15 min

(23, 30, or 37ºC baths)

Vortex

Incubate 15 min

Vortex

BSA STANDARD CURVE tubes

(Part B)

Set up Dilutions (in triplicate):

2 mg/ml BSASstandard

(in plastic vial) + diluent (0.9% NaCl)

Vortex

Bradford Test 100 uL of sample (at Room Temp)

OR 100L of Standard Dilution

mixed with 5.0 ml BioRad Dye Reagent

Vortex and sit 5 min at R.T. (on table)

Read Absorbance (OD) at 595 nm on spectrophotometer

set SPEC to 100% T 0 Absorbance) with “tube E” of standard Curve

no Water bath

PROCEDURE OVERVIEW & COMMENTS The Chart to the right summarizes the specific steps in the procedures you will carry out today

 ONE PART OF your team should start on Part A ENZYME DIGESTION EXPERIMENT, and finish with running these resulting samples through Part C, the Bradford Test.

 OTHER PART of your team should start on the Part B STANDARD CURVE DILUTIONS, and finish with running these resulting series of dilutions through Part C, the Coomassie Reaction. These data will be used to construct a Standard curve on the computer.

EXPERIMENTAL DESIGN and a data table for you to record your data are in the table 3 on following pages. Note the following:

 The total reaction volume is 5 ml in each tube. If substrate, buffer, or enzyme is omitted it is replaced it with an equal volume of diluent (0.9% NaCl, saline).

 For temperature effects, the options are room (23 ºC) warm (30 ºC) and hot (37 ºC)

 Teams testing pH effects incubate all sample tubes at 37 ºC; select 3 pH levels to test.

 Teams testing temperature effects use pH 3 for all sample tubes.

 Each treatment group in the experiment, and each concentration for the Standard Curve, is run in triplicate.

General Comments:

 Care is needed to avoid pipette- tip contamination.

 For each type of solution, when being added to test tubes, dispense in specific location (relative to the “front” where your label is) on the test-tube wall (keeping it in the test-tube rack instead of picking it up), to avoid cross contamination (when dispensing a stock solution into a series of tubes and reusing the same tip).

 Greatest accuracy may be obtained if, from a sitting position, you hold up the source container (e.g. flask of enzyme) to about eye level so that you can see the pipette tip as you immerse it just (1 mm) under the surface of the solution, and you can watch for bubbles, which occur when you do not control the rate of movement of the plunger on the micro pipettor.

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BIO 330L General Physiology 2015 Page 5

Details:

PART A. DIGESTION EXPERIMENT:  The tables on Page 9 (pH experiment) and Page 10 (for Temperature experiment) are

useful to use when pipetting.

 Check off when you have completed a particular item (e.g. adding pH buffer to all tubes requiring or diluent to all tubes getting the 0.9% NaCl “diluent”)

 TEMPERATURE TEAMS: Calculate what amount ot pH buffer (pH3) you need and dispense 1-2 ml more into a 50 ml flask (up at FRONT TABLE), to take back to your table.

 PH HYPOTHESIS TEAMS: Calculate what vol of each pH buffer you need and dispense 1-2 ml more than into a LABELED larger test tube in a rack at FRONT TABLE, to take back to your table.

AT YOUR TABLE: 1. Label your incubation test-tubes (in triplicate). Use a black or blue sharpie and write at the

top so the writing will not be immersed in the water baths during incubation. It will rub off when exposed to steam and then handled.

2. Dispense 2.00 ml of the correct pH BUFFERS into your empty, labeled tubes (as indicate on

the Protocol Table). Use gloves (pH 3 is quite acidic)

3. Next, pipette 1.00 ml (1000 uL) of protein SUBSTRATE (20 mg/ml BSA) to all tubes

designated to have substrate.

4. Add appropriate vol DILUENT (0.9% saline) to any tubes assigned this. It is used in any tubes

when enzyme or when substrate are omitted. Note that final total volume in any tube will be 5 ml, at end of set up (after enzyme is added last).

5. Add 2.00 mL ENZYME (5% pepsin) as indicated in your experimental design table, to any tube

assigned enzyme. Adding the enzyme last is important, since tubes should have close to identical time exposed to the enzyme. ALWAYS ADD ENZYME LAST!!!!

6. Cover each tube tightly with parafilm and vortex each tube. Hold (firmly) at the very top of

the test tube, between two fingers, and push down firmly until the entire content form a vigorous vortex (“whirlpool’ in the center from top to bottom). To keep parafilm on the tubes during incubation, be sure it is slightly stretched and that excess is wrapped around tube. “Heat-seal” the parfilm to the glass by holding several seconds tightly in your finger tips.

7. Place your tubes into the plastic test-tube racks in the incubation water baths. Incubate at

appropriate temperature for a TOTAL of 30 minutes, mixing once in the middle at 15 min. Do NOT vortex wet test-tubes--- Wipe with Kimwipe or paper towel first.

8. During the incubation of your digestion tubes, label a second set of short test tubes, with the

same ID’s as your incubating (digestion) tubes.

 This second set is for the Bradford-Test (see PART C, below).

 You will run the Bradford at the end of the incubations, after samples have cooled down to room temperature (RT).

 The solution temperature is very important for the Bradford test, because this will dramatically affect color (hence absorbance) if not at RT.

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PART B. STANDARD CURVE FOR BSA 1. Diluting the BSA (Albumin) 2 mg Standard for the standard curve.

 Following Table 1, prepare a set of protein standards, in triplicate.

 Dilute the albumin (BSA) standard (2 mg/ml BSA), which comes in a small glass ampule, with diluent- the same used for the samples (i.e. 0.9% NaCl).

 Break the top of the ampule at the narrowest point, holding it in a folded piece of Kimwipe.

 You have a enough to make a triplicate set of each dilution for the Standard Curve.

 Label your vials A1, A2, A3, etc., before starting.

From ampule

Volume Volume of Final of 0.9%NaCl BSA Stock BSA Vial (triplicates) Diluent Standard Concentration

A1 100 μl 300 μl 1,500 μg/ml = 1.50 mg/ml

A2 “ “ “” “ “ “ “

A3 “ “ “” “ “ “ “

B1 200 μl 200 μl 1,000 μg/ml = 1.00 mg/ml

B2 “ “ “” “ “ “ “

B3 “ “ “” “ “ “ “

C1 300 ul 100 ul 500 ug/ml = 0.50 mg/ml

C2 “ “ “” “ “ “ “

C3 “ “ “” “ “ “ “

D1 350 ul 50 ul 250 ug/ml = 0.25 mg/ml

D2 “ “ “” “ “ “ “

D3 “ “ “” “ “ “ “

*E 400 μl 0 ul 0.0 ug/ml = 0.00 mg/m = BLANK

* Vial E is the Standard Blank

2. Label another 12 of the 13x100 (small) test tubes for the A,B,C,D triplicates. These are so you can run the Bradford reaction for these standard curve test-tubes. This is described on the next page in Table 2.

Table 1. Preparation of Diluted Albumin (BSA) Standards Dilution. Scheme for Standard Test Tube Protocols using the STOCK ampule of 2 mg/ml BSA.

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BIO 330L General Physiology 2015 Page 7

 Be sure spectrophotometer is on and set the wavelength for maximum absorption of the Reagent (595 nm). Allow the spectrophotometer to warm up for at least 10 min.

 Standardize the spec with a test-tube the BLANK from the standard curve (the 0 mg/ml tube) in a square polystyrene cuvette, to adjust machine to 100% transmittance (0% absorbance). Follow the Vernier Instrument instructions.

. O.D.

Tube mg/ml rep 1 rep 2 rep3 Average

A 1.50

B 1.00

C 0.50

D 0.25

E 0.00

blank

Use this tube to set 100% transmittance

(0 Absorbance)

3. Generating the BSA standard curve.

a). For each triplicate, assess values and determine if there is a value that is a likely outlier (if the color looked different from the other two, to your eye, this is one good sign).

b) Plot the mean Blank-corrected 595 nm measurement for each BSA standard vs. its

concentration in μg/ml (or mg/ ml), and use “Custom SE” in Excel for each mean (entering the values you calculated for each).

c) Add a trendline (use 2

nd order polynomial- is nonlinear above 0.9 mg/ml).

d) Add X & Y gridlines (like graph paper), by selecting the Graph, then “Chart Options”, and

“Gridlines”, then both x and y gridlines. Decide how often (the more frequent, the higher accuracy when reading your curve to determine concentrations of the incubated tubes).

 You will need to HAVE your BSA curve to use, for your digestion samples, today. You don not have to solve the equatin, yoy may simply eyeball from the cirve, which is accurate if you print with the X and Y Gridlines.

 You must determine if any of your unknowns (digestion samples) need to be diluted and rerun through the Bradford (5 min); i.e. if the OD is too high (it will read 1._ on the SPEC or they may be “off the curve”).

 See bottom of page 8 for more information.

TABLE 2. Data for Standard Curve Triplicates: 1. Add 5.0 ml of Bio-Rad Reagent to 100uL of the standard curve dilution (each triplicate),

vortexing and holding at RT for 5 min. 2. Use the Blank (tube E having only diluents, i.e. 0 mg/ml BSA) to set spec at 100%

transmittance (0 Absorbance) with machine wavelength set at 595 nm. 3. Use the plastic transfer pipette (with squeeze bulb top) to put sufficient volume of each

triplicate of your unknown digestion samples into square cuvettes. 4. Measure OD (at 595 nm) of each triplicate tube A,B,C,D and record in table below

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BIO 330L General Physiology 2015 Page 8

PART C. BRADFORD-Protein Assay with the BIO-RAD REAGENT:

 Be sure spectrophotometer is on and set the wavelength for maximum absorption of the Reagent (595 nm). Allow the spectrophotometer to warm up for at least 20 min.

 Read the instructions for the Spectrophotometer.

 Standardize the spec with a test-tube the BLANK from the standard curve (the 0 mg/ml tube) in a square cuvette to adjust machine to 100% transmittance (0% absorbance).

 Solutions (including samples) MUST BE at room temperature before starting the Bradford test. You should immerse rack in a slurry of ice water but do not let it get too cold. After 1-2 minutes, hold a test tube and measure the temperature of the contents. You only need 100 ul of each sample this volume. Pipetting is temperature-sensitive, so your solution must also be at RT to pipette this 100 uL volume accurately.

Running the Bradford Protein test when solutions are at RT (room temperature):

a. First add 100uL of the unknown sample triplicate to each empty labeled empty small (13 x100) test-tube. Use a new pipette tip for each sample

b. Add 5.0 ml of the BIO-Rad Bradford Reagent

c. Vortex and let sit 5 minutes at RT.

d. Use the plastic transfer pipette (with squeeze bulb top) to put sufficient volume of the each

triplicate of your unknown digestion samples into a square cuvette. Use a new pipette for each sample.

e. Read the OD (absorbance) of each tube on the spectrophotometer at 595 nm.

f. Use the BSA standard curve (Part B) to determine the protein concentration of each unknown digestion sample (Part A).

*** NOTE: You must RETEST Any incubated sample that is out-of-range on the HIGH end (more concentrated, which will read 1._ on the Spectrophotometer). First try diluting in half: mix 100 uL of original incubated sample + 100 uL diluent, vortex, then take a 100 uL sample of this and add 5.00 ml Bradford dye. Wait 5 min and measure the OD from this tube, determine the concentration using the standard curve. T Then to calculate the original concentration double this value (dilution factor was 2, so you multiply by the dilution factor).

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SET-up and DATA TABLE for pH Hypothesis

EXPERIMENTAL SET UP: diluent (0.9% saline) RESULTS Tube ID #

Incubation

Temp

Substrate BSA at

20 mg / ml

Buffer

pH

Enzyme

5 % Pepsin (or NaCl diluent)

notes

O.D.

mg / mL protein conc.

off Std Crv

Calculated

mg protein

remaining

Calculated

mg protein digested

1a 37 °C 1.0 ml BSA 2.0 ml pH 3 2 ml enzyme

1b 37 °C 1.0 ml BSA 2.0 ml pH 3 2 ml enzyme

1c 37 °C 1.0 ml BSA 2.0 ml pH 3 2 ml enzyme

2a 37 °C 1.0 ml BSA 2.0 ml pH 3 2 ml DILUENT

2b 37 °C 1.0 ml BSA 2.0 ml pH 3 2 ml DILUENT

2c 37 °C 1.0 ml BSA 2.0 ml pH 3 2 ml DILUENT

3a 37 °C 1.0 ml BSA 2.0 ml pH 5 2 ml enzyme

3b 37 °C 1.0 ml BSA 2.0 ml pH 5 2 ml enzyme

3c 37 °C 1.0 ml BSA 2.0 ml pH 5 2 ml enzyme

4a 37 °C 1.0 ml BSA 2.0 ml pH 5 2 ml DILUENT

4b 37 °C 1.0 ml BSA 2.0 ml pH 5 2 ml DILUENT

4c 37 °C 1.0 ml BSA 2.0 ml pH 5 2 ml DILUENT

5a 37 °C 1.0 ml BSA 2.0 ml pH 7 2 ml enzyme

5b 37 °C 1.0 ml BSA 2.0 ml pH 7 2 ml enzyme

5c 37 °C 1.0 ml BSA 2.0 ml pH 7 2 ml enzyme

6a 37 °C 1.0 ml BSA 2.0 ml pH 7 2 ml DILUENT

6b 37 °C 1.0 ml BSA 2.0 ml pH 7 2 ml DILUENT

6c 37 °C 1.0 ml BSA 2.0 ml pH 7 2 ml DILUENT

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BIO 330L General Physiology 2015 Page 10

SET-up and DATA TABLE for TEMPERATURE Hypothesis

EXPERIMENTAL SET UP: diluent (0.9% saline) RESULTS Tube ID #

Incubation Temp

Substrate BSAlbumin

20 mg / ml

Buffer

pH

Enzyme

5 % Pepsin (or NaCl diluent)

notes

O.D.

mg / mL protein conc.

off Std Crv

Calculated

mg protein

Remaining

Calculated

mg protein

Digested

1a 23 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

1b 23 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

1c 23 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

2a 23 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

2b 23 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

2c 23 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

3a 30 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

3b 30 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

3c 30 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

4a 30 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

4b 30 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

4c 30 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

5a 37 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

5b 37 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

5c 37 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml enzyme

6a 37 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

6b 37 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

6c 37 ºC 1.0 ml BSA 2.0 ml pH 4 2 ml DILUENT

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GLASSWARE CLEAN-UP Sinks with Soap buckets stations are located by the sinks on far wall (west bench). TEST TUBES

 Small tubes (Bradford and std curve tubes): Discard contents down sink. Throw tube in GLASS discard BOX.

 LARGER TEST TUBE (solutions or for INCUBATIONS): WASH with soap, scrub off label, invert clean tubes in test tube rack (if open bottom) or in square wire baskets and return to your lab bench tray.

SMALL FLASKS  Dump contents down sink, rinse 3-5 times with water. Tap upside down on some paper towels,

return to your tray.

 Leave inverted on paper towels on your metal tray at your table.

PIPETTES

 Most are Disposable (will be indicated on the pipette)- then put in GLASS box (NOT regular garbage).

PLASTIC PIPETTE Tip Box: Refill the two tip boxes on your table for the next lab. The bags of tips are on the front table.

ENZYME Data ANALYSIS for your Assignment due Next Week

There is a separate file containing the Grading Rubric, and the Template to use for the DATA SHEET” assignment for this lab. This template presents a series of questions, asks to show your calculation and graphs. The following gives you some instructions on analysis and graphs. Data Evaluation:

1. Evaluate each set of triplicates, and decide if there is a really “odd-ball” value. You may elect to not use a tube if it is REALLY different from the other two. But the other two need to be fairly close together to decide that the third is an outlier. You may calculate the average of the 2 (although not statistically valid to do so). This is why we do triplicates. This is especially important for the standard curve (i.e. you may through one triplicate point out if it makes your curve awful, because you DO know what it should look like).

Calculations:

1. Use the standard curve to determine the concentration of protein in your unknown

(incubated) samples. You may simple eyeball the value IF you have printed graph (grid) lines on your Excel graph). Calculate the Average of each triplicate. This value is the amount protein remaining after the incubation period.

2. Calculate and graph the amount of protein DIGESTED (not amount remaining, which is what you have measured !!!!). a. First, calculate the amount (in mg) of BSA protein that you started with. The initial substrate

concentration is 10 mg/ml. So you had 10 mg to start with (since you used 1 ml of this). If a final concentration after digestion is 1 mg/ml, then you need to calculate how many mg total must have be in the incubated tube (which had a total vol of 5 ml). Show a sample calculation in your ASSIGNMENT. The difference between starting and ending mg of protein is the amount digested.

b. For your data set (pH or temperature) calculate the mean and the SE of the mean (review "Topic 1 GRAPHS STATS Lab) amount digested.

4. Plot (graph) the mean (± 1 SE) amount of protein DIGESTED as a function Temperature or

pH depending on your experiment. Fully label the graph (as detailed Topic 1 exercises).