Cell Physiology Lab 2004 Mary Lee Sparling This is a list of possible experiments. We may change the order according to availability of material, or we may decide to do individual projects in place of some of these.

Feb 2 INTRODUCTION-what do we want out of this lab? How well do we make solutions?

4 PROTEIN TEST-PREPARE STANDARD CURVES FOR USE ALL SEMESTER

9 PHOSPHATE TEST

11 FERTILIZATION OF SEA URCHIN EGGS AND DRUGS WHICH ARTIFICIALLY ACTIVATE OR PREVENT FERTILIZATION

13 Last day to drop class without approval

16 MEMBRANE PHOSPHOLIPASES AS DETECTED BY TLC

18 SECRETION, ENDOCYTOSIS

20 last day to add class

23 CELL MOTILITY;SLIDING FILAMENTS:TRADESCANTIA AND ELODEA

25 MEMBRANE Preparation, use of detergents

March 1 ACETYLCHOLINESTERASE

3 CILIA AND FLAGELLA, motors and microtubules

8 turn in lab books, try to set up microelectrodes setup

10 INHIBITORY SYNAPSES AND DRUGS SPECIFICITY AT DIFFERENT SYNAPSES

15 GLYCOLYTIC ENZYME CONTROL BY HORMONES AND ENERGY CHARGE

17 PURIFICATION OF TUBULIN FROM CHICK BRAIN

22 PCR or DETECTION OF MRNA

24 DRUGS WHICH EFFECT DIVISION

29 MEIOSIS BLOCKS IN EGGS AND THEIR RELEASE TO COMPLETE MEIOSIS

31 holiday

April 5-10 spring break

12 FISH SCALE PIGMENT MOTILITY

14 PURIFICATION OF MYOSIN AND ACTIN. TURN IN LAB BOOKS.

19 MYOSIN ATPASE

21 ELECTROPHORESIS

26 ELECTROPHORESIS STAIN AND INTERPRETATION

28 ANTIGEN-ANTIBODY INTERACTION- OUCHTERLONY AND ELISA

May 3 REGENERATION PLANERIA, EFFECT OF RETINOIC ACID, DRUGS

5 CELL INTERACTIONS- LONGTERM-MORPHOGENS, INDUCTION

10 APOPTOSIS CONTROL AND PREVENTION

12 ACTIN ASSEMBLY

17 CANCER, WHAT CONTROL IS LOST IN ONCOGENES?

19 last day-CLEAN UP LAB AND FINISH LAB BOOKS Prepare poster of best experiments.

580 LAB INTRODUCTION

1. We can assemble some systems to try working with artificial membranes and single cell microelectrodes.

2. We can purify some proteins. We can use centrifugation, columns, and salt extraction. (myosin, tubulin, actin, Na+K+ATPase)

3. We can do electrophoresis and Western blot with antibody detection of proteins

4. We can observe cell motility, organelle motility in cells and effects of drugs and salts.

5. We can do membrane lipid purification and analysis

6. We can study cell cycle and drugs which modify it in sea urchin eggs. We can do immunohistochemical detection with antibodies.

7. We can study protein kinases and phosphatases.

8. We can do protein and phosphate tests and standard curves.

THE WAY THINGS ARE CHANGED IN CELL PHYSIOLOGY LAB

We used to do experiments where we did not know how they were supposed to come out, then we tried to explain our results. We were given the hypothesis to test, we were given the chemicals, and we were given directions of how to do an experiment.

Now we are given a topic, let's say "How does cytoplasm move? " We are given an idea of available equipment, reagents, animals or plants. Then we are told- observe something about cell movement or at least something to do with cytoskeleton.

Decide how a certain system can be used to test an hypothesis: for example,

1. A sea urchin sperm can only enter an egg if both of them can polymerize an available supply of G-actin. Then you can test various ways to prevent actin polymerization to see if it prevents fertilization. You would have to do a library search using let's say: su:actin polymerization fertilization prevention or acrosome pH Calcium osmotic pressure. So instead of figuring out what 1M glycerol does, you would figure out what it might do before you use it- why do you use that instead of salt?

2. pollen cannot germinate without the assembly of actin ( test in the presence of cytochalasin or colchicine or metabolic inhibitors.)

3. pollen cannot germinate without first altering the pollen coat, and the continual softening of the advancing cell wall tip (in the presence of a protease inhibitor or a cellulase inhibitor or a protein kinase inhibitor or EDTA)

4. Elodea chloroplasts can move around on an actin sheet in the presence of ATP and Ca++. Then you have to figure out how to cover a slide with actin filaments.

WHAT TO USE IF YOU WANT TO TEST THE EFFECT OF SALT

Substitute equal osmotic material that does not ionize- glycerol, sucrose, urea, ficol.

Change the concentration, or substitute divalent for monvalent, or K or Li for Na.

Add EDTA or EGTD to tie up divalent cations.

Use sephadex to change ionic medium.

Use ion exchange chromatography and elute with different salts to see which has an effect.

Use ionophores.

Change ratio of monovalent and divalent ions.

Precipitate with ammonium sulfate- salting out.

Precipitate by dilution- myosin.

HOW TO TEST FOR INVOLVEMENT OF CA++

Use A23187.

Add EDTA or EGTA or citrate to chelate it.

Inject it.

Add Ca++ pump poison.

Poison Ca++ channels

WHAT TO DO TO TEST THE EFFECT OF METABOLISM

Add inhibitors of glycolysis or CAC or ETS.

Cut off O2 supply.

Cut off CO2 supply for plants

Remove light for plants.

Remove food.

Uncouple oxidative phosphorylation

HOW TO TEST THE EFFECT OF WATER- use heavy water or carbowax to cut down amount of water in cell.

Use glycerol to cut down water concentration.

HOW TO TEST EFFECT OF HYDROGEN BONDS

Break bonds with urea or heat. With actin and tubulin, depolymerize with cold.

FIND A SPECIFIC DRUG THAT PREVENTS AN ACTION Then try different concentrations or time of application.

To TEST FOR EFFECT OF PH. Carefully prepare buffers of different pH, or apply materials which alter internal pH like ammonium chloride.

Use amiloride to prevent H/Na exchange

HOW TO TEST FOR ACTION OF ATP OR G PROTEIN OR SECOND MESSENGERS:

Look up specific inhibitors of these in reactions. There is a handbook on inhibitors in the library. Vanadate effects ATP-utilizing proteins , inhibits phosphatase.

HOW TO TEST FOR EFFECTS OF PHOSPHORYLATION

Test for increases or decreases of P-amino acids.

Test effects of cAMP, cGMP, ATP, GTP in presence of permeator molecule such as digitonin.

Lead can trap phosphate broken off molecules, then be made black to observe.

TO TEST FOR MEMBRANE INVOLVEMENT

Use detergents, Triton-X, digitonin, ionophores.

Extract with chloroform-methanol.

Use centrifugation to pellet membranes to see if material soluble at certain times, attached at others.

Make membrane vesicles, isolate, turn inside out, see effect.

Teat for endocytosis of material, exocytosis prevention or initiation.

Get temperature effects due to lipid phase change.

HOW TO TEST FOR ACTION OF MEMBRANE POTENTIAL

Change salt gradients. Add channel poisons, change charge across membrane with voltage clamp.

HOW TO TEST FOR NUCLEAR INVOLVEMENT

Add actinomycinD to prevent RNA synthesis, puromycin to prevent protein synthesis

TEST FOR ENZYME INVOLVEMENT

Use many poisons, one at a time.

Precipitate out protein with TCA,

Test Q10 of reaction to see if just diffusion or if enzyme.\

HOW TO TEST FOR -SH INVOLVEMENT OR REDOX POTENTIAL

Use DTT, mercaptoethanol, diamide,

TECHNIQUES USED TO TEST CHANGE

centrifugation

thin layer chromatography

gas-liquid chromatography

electrophoresis

observation in microscope

antibody reaction for immunocytochemistry or gel detection

immunodiffusion plates

enzyme assay

spectrophotometric detection by wavelength

column chromatography

gel filtration

microelectrodes, voltage clamp

cell injection

image analysis

fixation, sectioning and staining

dialysis

salting out

alcohol precipitation

lipid extraction

fluorescence detection

drug treatment

milipore filtration

detergent extraction

high salt extraction

microdissection

isoelectric focusing

recombinant dna

reporter genes

make models or cell-free systems such as tubulin or actin on a slide with motors moving over them

affinity chromatography

hydrophobic chromatography

cell ph or ion concetration detection by dyes or microelectrodes specific to one ion

fluorescent or other analogue chemistry

TO PURIFY A PROTEIN, need a way to follow it through the procedure and to detect presence of contaminants:

enzyme assay, electrophoresis, antibody, radioactivity

INDIVIDUAL PROJECTS CAN BE SUBSTITUTED FOR STANDARD LAB WORK.

INTRODUCTION

This cell physiology lab is designed to do several things:

1. Acquaint you with biological experimentation as a non-exact science

2. reinforce your knowledge of the experimental method, force you to start out each experiment with an hypothesis to test, make you aware of the importance of control experiments for comparison;

3. Allow you to think of cell activities as products of control of the cytoskeleton, secretory and endocytotic mechanisms, and metabolic machinery which can result from nuclear or environmental stimuli which produce alterations in membrane structure or other changes which can result in ionic concentration differences or enzymatic alterations. Our most common experimental variables will be temperature, calcium and magnesium ion concentration, cytoskeletal disruptors, metabolic inhibitors and substrate concentration, and anesthetics that alter the membrane.

4. Allow you to learn some of the methods used in biological research in cell physiology. This will be a great advantage to those going into graduate work, or teaching, or professional school. Not very many universities have courses in cell physiology. That is why I had to write this manual, so you are fortunate to be able to have this opportunity as an undergraduate.

Occasionally we have labs which do not produce the expected results of the hypothesis. The common error of students is to think that these labs failed. It is just as important to your education in biological research to figure out why some experiments do not "work out" the way they were expected, as it is to get beautiful results. Most of research is spent getting the experimental conditions to the point where data can be collected and meaningful results collected. Research is not cookbook chemistry, and I have not attempted to write a cookbook. I have taken some experiments from the literature and tried to adapt them to classroom application. There are often time constraints, which make this difficult. Sometimes, we try a different species due to availability. Sometimes experimental animals die right before the class, sometimes mistakes in solution making can occur, since graduate students often are also doing this for the first time, and sometimes the lab temperature is very warm or very cold which can change rates of egg development, or cell motility.

No lab should be considered wasted, since you will learn to handle volumetric measurement, microscopic proficiency, quantitation, methods for handling proteins, enzymes, live cells. If you are consistently getting nonsensical results when everyone else is not, then you must ask for help. You may have bad pipetting methods, solution labeling methods, glassware washing methods, or data recording technique.

You can help insure good lab technique by the following:

1. Purchase a glass marking pen, some graph paper, a cheap stopwatch, and keep them in your backpack.

2. Take good notes during the experiment about any variation of technique and record your results accurately. You can save yourself enormous amounts of lab time by setting up tables ahead of time for filling in the results.

3. Read about the experiment in your text and any references in the lab manual before you come to the lab. You will get much more out of the experiment. You should write some of the possible conclusions to such experiments before you do them. For example, if you are going to vary the ion concentration in a certain enzyme test, find out what that ion does in cells, in relation to that enzyme so that you will have a possible explanation of your results. You will not be able to figure out what your experiments mean unless you do this. You may want to bring your texts to class as resource books, or your biochemistry text may also help. If the experiment involves cytoskeletal disruptors, find out what each one does, and write it in your lab book, since that can be referred to many times. Since you always have to turn in your lab results at the end of the week, you must have some of the work done before the lab.

4. Don’t rely on the instructor to tell you what this experiment is supposed to mean. I will be happy to answer questions, but I expect you to do some work before you come. It is not always possible to coordinate the lecture with the lab, so sometimes you will do something in lab we have not yet discussed in lecture. Therefore, you will have to read in your text to find out where it fits in the overall cell physiology. That reading will just make it easier when we come to that part in lecture.

5. Always use clean glassware. Most of the time you can see dirt in glassware. You will not have a problem if you always leave it clean at the end of the lab, to dry for the next time. Always take clean pipettes from stock for the day, and then put them back at sink to be washed at the end of the day. Glassware should be immersed in hot soapy water and brushed individually, then rinsed eight times in running tap water and three times in distilled water. Invert to dry. Disposable pipette tips and cover glasses need not be washed.

6. Keep your lab exercises after they have been graded. Your lab book will be handed in at the end of the semester for review and grade. The lab assistant will grade some experiments, but the instructor will make the final analysis. Do work with your lab partners, but write up your experiments by yourself, since they will be compared at the end. That is where the prelab work will show up. Don’t copy material from books or articles without referencing them. Looking topics up on the web of on your text CD is also a great idea.

SOME LAB TIPS

PIPETTING. For good control of the pipette use your index finger on the tip of the pipette, not your thumb if using the blue bulbs. Pull fluid up into the pipette until slightly above the place where you want the volume to be. Release your BULB at the same time as you place you finger on the tip. If the tip of the pipette is wet, you will not be able to easily release a part of the volume to get it to the volume you wish to deliver, so don't have wet fingers. Probably the most accurate way to pipette is by the blowout method, where you only take up the amount you wish to release, each time you place a volume in a tube, then blow out the fluid using the bulb after it flows to a stop. With the black bulbs, always make sure you remove the cotton from the pipette, and for small volumes don’t have a high vacuum on the bulb or you will suck fluid into the bulb. Always use the size pipette near the volume you want (use a 1 ml for .1-1, use a 5ml for 1.1-5, a 10 ml for 5.1-10.0. Each time you pipette, you magnify the error. Don't use large pipettes for small amounts to deliver, because you cannot read the large diameter as accurately as the smaller diameter pipettes. For amounts less than .1 ml use the automatic pipettes with disposable tips. During the same lab, you can use the same tips, if you label them by marking them with a marker to prevent contamination of the reagents.

For all materials use rubber bulbs. Most lab chemicals are not toxic, but by law we cannot mouth pipette.

DILUTIONS. To make dilutions, divide the desired concentration by the present concentration, and then use that value to determine the desired volume of concentrated reagent to dilute with water to the final volume.

Example: reagent prepared is 1 M. Reagent concentration desired in your reaction mixture is 6 mM or .006M. Divide .006 by 1= .006. You need 25 ml of reaction mixture, so multiply 25 by .006= .15 ml you need to add before bringing the mixture to a final volume of 25 ml. Often in making reaction mixtures you have to add several reagents, so add up all the reagent volumes added, subtract from the 25 ml, then make up the difference by adding water. Bringing a calculator to lab is essential. IT IS IMPORTANT NOT TO DILUTE REAGENTS TO THE FINAL CONCENTRATION DESIRED BEFORE ADDING THEM TO THE REACTION MIXTURE BECAUSE THAT WOULD DILUTE THEM FURTHER.

pH. The pH of reactions is very important and must be controlled during experiments. Make sure that all reagents are the proper pH, and then add buffer at the controlled pH desired. Usually, use 0.01M or less final concentration.

SPECTROPHOTOMETERS. Always check to be sure the filter system is correct for the wavelength you wish to use. Then turn it on 20 min before you need to use it, so it can stabilize. Use only special tubes, and insert them so the markings are at the front. Make sure you know how to zero the instrument, and have a control tube for doing that.

WATER BATHS. Fill the water bath with water the approximate temperature you want, and then adjust it with ice and a thermometer. For heat, use warm water, then turn on the heating element and adjust it so the light just comes on at the desired temperature, and goes off when you turn it a little bit lower. Then watch it for a few minutes, to be sure it is right.

REAGENTS. Since the whole class has to use the reagents, it is important that dirty pipettes not contaminate them. Take the amount of reagent you have figured out that you need for the whole day, placing it in an Erlenmeyer flask, well marked. Since there are 16-20 students and 8-10 pairs of partners, never take more than 1/10 of the total volume. At the end of the day, you can put back reagent that you feel is uncontaminated, particularly expensive ones like ATP, cytoskeletal disruptors, protein standard reagents, phosphate standard reagents, GTP, buffer, sucrose. Always keep high-energy compounds like ATP, GTP, G6P, acetylcholine, or proteins isolated from cells on ice during the day since they break down at room temperature. Never add them to your reaction mix until the last minute.

ABSENCES. There will be two absences allowed, with library work to makeup labs. Habitual tardiness, or long coffee breaks, or allowing your lab partner to do the work, is always noted by the instructor and will be reflected in your final grade.

GRADING will be subjective. An A grade will be for those who do the work, write the reports, including drawing correct conclusions about the experiments. This will include getting results on unknown samples given by the instructor. To be able to do all of this in 3 hours takes organization, done largely before coming to lab. The instructor will do spot checks to see if you have done your prelab work. It is possible for everyone in lab to get an A, if everyone does the work well.

REFERENCES FOR LABORATORY EXPERIMENTS RESERVE ROOM SOUTH LIBRARY

UNDER SPARLING, BIO580L

Dynamic Models in Biochemistry. A workbook of computer simulations using electronic spreadsheets. D.E. Atkinson, S.G. Clarke, D.C. Rees. Benjamin/Cummings Publ. Co.

Mathematical Models in Plant Physiology. J.H.M. Thornley. Academic Press. 1976

Analytical Chemistry G.D. Christian 1986

Physical methods on biological membranes and their model systems. F. Conti, WE Blumberg, J. de Geir, F. Pocchiari . Plenum Press. 1982. Reaction

Quantitative Analysis by gas chromatography. J. Novak. M. Dekkar, N.Y. 1975.

Mathematical Models in Plant Physiology. JHM Thornley. Academic Press 1976.

Data reduction and error analysis for physical sciences. PR Bevington. McGraw Hill 1969.

GET NEW LIST FROM INSTRUCTOR

ERROR ANALYSIS FOR BIOLOGY 580 LAB I.

How can one evaluate the expected error for an experiment? Break up this answer into parts:

1. figure out the expected error for each step in the test See Analytical Chemistry by Christian.

A. Purity of chemicals: analytical reagents are 99.95% pure B. Weighing: the last digit of the scale gives the sensitivity analytical balance to .1 mg,

larger scale to .1 g or .01g (see scale)

so the accuracy depends upon the amount weighed since .1 g is a lot less of the total weight when you have 30g than when you have .3 g total.

C. Pipettes: blowout two rings around top

10 ml error .02

5 ml .01

1 ml .006

automatic pipettes to 1‑2% or .01-.02

D. Volumetric glassware:

100-1000 ml volumetric flask .0003

graduated cylinders .01

To calculate the expected error in your experiments you use these values X the total you are using (g,ml) and that squared gives you the variance for what you really measure out- expected error.

E. calculate the total error‑ all the errors combined, some of which may cancel out others by adding up all the variance (error squared.)

See Taylor, or Christian, or Bevington, or Freund

We will do this on a spreadsheet where you will enter

1) the proper expected error from the list above, and the amount measured (ml,g) and it will automatically give you the expected error which will then be squared to give you variance for that sample or solution in the columns so marked. Then in the end, you will get the total for each sample in the standard curve or enzyme analysis.

2) When you are doing a standard curve you have more than one sample, so you have to multiply the total variance by the number of samples to get the total variance for the test.

3) to get the expected error for the test, which includes getting the regression line and equation, you take the square root of the total variance.

What is the difference between accuracy and precision? The accuracy of a test is just what we were just talking about, but the precision is a matter of how repeatable your results are.

What is the difference between standard deviation and variance? How can variance be used to determine the total error in additive tests?

EQUIP

experr

amt-gorml

ERR

VARIANCE

NUM STEPS

NUM SAMP

phosphate

TCA

balance

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test

vol

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pipette

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po4

balance

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vol

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pipette

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water

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sulf-mo

vol

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gradcyl

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gradcyl

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bal

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