Euglena has a light spot at the anterior end at the base of the flagellum which has been implicated in phototactic responses. The eyespot is associated with the flagellum. The action spectrum of phototaxis corresponds to the absorbance of the eyespot with a peak at 480-500 between blue and aqua. Each group should apply different colored plastic to holes made as a vertical series of windows, (each with a different color) down the side of the test tube, where the fluid of the culture is in a black paper used to cover the sides of a test tube. Make sure no light can get into the tube except through the holes.Place lights within 20 inches of the tube and holes (don't get the tubes too close or hot, and look at the distribution of the organisms after 1-2 hrs. When making conclusions, remember that the color of something like the plastic shows what color is reflected, not sent through it. Do this by carefully decanting with the windows held up.

Effect of ions on movement

Euglena can switch to an amoeboid or more appropriately, a euglenoid movement under certain conditions since it has a rather soft pellicle. With the foillowing experiments, try to determine what those conditions are. In these experiments use bottled water, not tap or distilled water. Place 1 ml of culture for the whole class into 1 ml of the following 1/16 M solutions;

NaCl,KCl,MgCl2,CaCl2,Na2SO4,NaI. Then each group should observe the rate of movement in one drop on a slide. Then different groups can each try 2 mixtures of the above with one drop in each of two different ions. Give the euglena a chance to equilibrate in the new mixture, or observe as they do, noting the difference when a divalent cation is mixed with a monovalent and another divalent cation. Look up the effect of gibbs donnan equilibrium changes on ions bound to microtubules and microfilaments due to such mixtures.

bound monovalent cation = free monovalent cation ----------------------- ---------------------- bound divalent cation SQUARE ROOT OF free divalent cation

tHIS SAYS THE RATIO OF BOUND MONO TO DI-VALENT CATIONS EQUALS THE RATIO OF FREE MONO TO SQUARE ROOT OF FREE DIVALENT CATIONS

Can you get recovery of stopped ones by adding a drop of water? Observe for at least 5 minutes after dilution. Looking at the equation, you can see that dilution with water does not simply change this value in the equation, since the square root of the divalent ion concentration does not change at the same rate as the monovalent ions, by dilution.

Effects of pH on motility observe motility using a drop of culture and a drop of buffers ranging from 5.5-9. Each group works alone on this.

Effects of alcohol on motility. Each group should do this alone. To .5 ml of culture, add drops one at a time of 2% alcohol solutions, wait 2 minutes and observe movement after each drop until it stops. If it stops with one drop, dilute the alcohol 1/10 and start again. Use all of these alcohols which differ in chain length, and therefore rate of penetration of the membrane: methyl,ethyl,propyl,butyl,amyl. See if they recover after dilution with water. Keep in mind when writing up your results that the cell membrane regulates intracellular ion concentrations important in motility, and that motility occurs due to microtubules or microfilaments and other associated proteins.

ACTIN POLYMERIZATION AS DETECTED BY DNAase INHIBITION

References: Cell 15:935-943. 1978; J. Biochem. 87:785-802. 1980.

Stock solutions: 1. DNAase I- .1 mg/ml in .5 mM CaCl2, 50 mM TrisHCL pH 7.5 prepare at the time of the lab by adding solvent. 2. DNA (calf thymus) cut DNA fibers and dissolve by stirring overnight after homogenizing in a glass homogenizer in .1 M tris pH7.5, 4 mM MgSO4, 1.8 mM CaCl2. Measure absorbance at 260 nm, should be .5-.65 OD. This should be 1 mg/ml, dilute 1/25. 3. actin extracted from acetone muscle powder 4. guanidineHCL- 1.5 M GuHCL in 1 M sodium acetate, 1 mM CaCl2, 20 mM trisHCL pH 7.5

Equipment: quartz cuvettes, micropipettes-10,15,30 ul; UV spectrophotometer Quartz cuvettes should only be cleaned with Q-tip cotton swabs, soap, distilled water. Handle with care- they cost $30 each.

Purpose: to make a standard curve of inhibition of DNAase by G-actin, then to use that to detect how much G- or F-actin is present in an unknown (later we will use sea urchin eggs before, during and after cleavage)

Step 1. detection of DNAase without inhibition. This is done by mixing 10ul of DNAase with 3 ml DNA (solutions above) and immediately observing OD at 260 nm over a period of 5 min. We will have to use the spectrophotometer in the adjacent room. It holds four cuvettes, so several can be done at once from the different groups by reading in series. Plot the results and get a slope.

Step 2. detection of g-actin inhibition using known amounts of G-actin. Run a Biorad on your actin sample. Then prepare the following: (.5 ml each)

1. 20 ug/ml

2. 100 ug/ml

3. 300 ug/ml

4. 400 ug/ml

5. 600 ug/ml (if your actin is not this concentrated, make a similar but more dilute series.)

Now place 15-30 ul of the actin into a cuvette,( always use the same volume in your tests from then on) with 10 ul DNAase, stir immediately to mix the actin and enzyme and quickly add 3 ml DNA (have all these things ready) and observe in spectrophotometer and plot, get slope, and determine the amount of inhibition by the actin. Do this for each concentration of actin. Plot amount of inhibition (%) vs. actin concentration. Complete inhibition of 1ug DNAase by 1.35 ug actin according to the literature. The lower limit of detection of actin should be 10 ug/ml actin, upper limit 1.8 mg/ml.

Step 3. Now prepare some F-actin (someone else can be doing this ahead of time) by adding .5 ml 2M KCl to 4.5 ml G-actin. (This can then be used for viscometry). Save .5 ml for use in this test, using it about an hour after adding the salt. Dilute the F-actin, using a concentration of actin which gave at least 50% inhibition of DNAase, use the volume used previously with G-actin, doing an inhibition test (this will not inhibit as much because much of the actin will be in the F-form). Now take a volume of the F-actin (like .1 ml) and add the same volume of guanidineHCl and incubate at 0" for 5 min. Then take 2x the volume (of this treated actin) usually used in the inhibition test (since you diluted with GuHCl) and do the DNAase inhibition. This will give you the value of the total actin present, since the guanidine causes depolymerization. Subtract the amount of G-actin in the test without quanidine from the value in the test with guanidine, and that is the amount of F-actin present. Now we can use this test on extracts of cells where there is not enough actin to extract and do viscometry. Hand in the inhibition standard curve and the values for G-actin and F-actin and total actin in your polymerized actin.

VISCOSITY MEASUREMENTS OF ACTIN POLYMERIZATION TO F ACTIN From the protein readings you took on the actin, figure out the mg/ml. If you have 5 mg/ml, for the experiments below, you would take 0.5 ml and dilute with 4.5 ml tris-ATP-CaCl2 to give Actin at a final concentration of .5 mg/ml in .01 M TRIS pH 8,10-4M ATP, 10-4 M CaCl2. Actin can be induced to polymerize by addition of 2.5 M KCl to give a final concentration of 50mM. DO NOT ADD THE KCl UNTIL YOU HAVE TAKEN A VISCOSITY MEASUREMENT OF THE DILUTED G-ACTIN WITH EVERYTHING ELSE, BUT WITHOUT THE KCl. PLACE 5 ML IN THE VISCOMETER AFTER HEATING IT TO ROOM TEMPERATURE BY IMMERSING IT'S CONTAINER IN TAP WATER AT 25 DEGREES. USE A STOP WATCH TO SEE HOW MUCH TIME ELASPED BETWEEN WHEN THE ACTIN PASSES THE TOP CROSS MARK TO WHEN IT PASSES THE BOTTOM MARK. REPEAT, AND AVERAGE THE RESULT. Then add the amount of KCl to polymerize the 5 cc of actin (.05/2.5= 1/50 dilution or 0.1/5, so add 0.1 ml 2.5 M KCl to the actin in the bulb of the viscometer, ROTATE to mix, RECORD THE TIME, and do a viscosity measurement by reading the number of seconds for the actin to traverse the distance between the two cross marks and record it. Do that every 2 minutes or however long it takes to run the volume through in case that is more than two minutes. Stir the actin in the bulb between readings by rotating the viscometer. Keep the readings going for 15 minutes. Now try to see if actin will polymerize in the presence of an oxidizing agent. Prepare 5 ml of G-actin as above but with diamide in it. That means you will add to 0.5 ml actin (or whatever volume in your case gives 2.5 mg actin)+ whatever volume of tris-ATP-CaCl2 to make up to 5 ml, plus diamide at a concentration given by your instructor. Measure viscosity and add KCl as before and follow viscosity change. MAKE SURE ALL SOLUTIONS ARE AT ROOM TEMPERATURE WHEN YOU START TO MEASURE THEIR VISCOSITY. ACTOMYOSIN VISCOSITY Prepare the actomyosin by adding some unfrozen myosin (1/5 the concentration of the actin) to a diluted 5 ml preparation. Try to have a concentration which will give an initial flow time at room temperature of 2-4 minutes, using 5 ml in the Ostwald viscometer. figure out the protein concentration in mg/ml from your protein tests and the dilution. After determining this flow time, add 0.05 ml 0.1 m ATP and 0.05 ml of 0.01M MgCl2. Keep doing readings until it reaches a steady state (this may take 40 minutes.) There should be an initial decrease and then a rise back to what it was when you started. Plot a graph of flow time versus time after addition of reagents. Try this at 20,25, and 30 degrees and calculate the Q10. What does this show about the reaction? Is the Q10 the same for the increase as for the decrease? (Remember in trying to explain the results that there is 10-4M ATP in the actin.)

EGG CORTEX ISOLATION

Unfertilized or fertilized eggs prepared according to the fertilization exercise are washed after settling in 5 volumes of 0.1 M MgCl2, 1 mM Tris pH 8 to stabilize the cell membranes and cortex. They are centrifuged at 5000 rpm for 5 min, and homogenized in 10 mM MgCl2- 1 mMTris pH 8. The cortices are sedimented again at the same rate and resuspended in the same medium and rehomogenized, and this is again repeated. The final pellet contains the ghosts or cortical hulls. These hulls can then ge used for the extraction of actin, tubulin or the assay for enzymes such as G6PD, Na+K+ATPase.

NA+K+ATPASE FROM MEMBRANES OF SEA URCHIN EGGS

OBTAIN EGGS, FERTILIZE THEM, AND PREPARE CORTICAL HULLS AS IN OTHER EXPERIMENTS.

ASSAY THE ATPASE WITH THE FOLLOWING REACTION MIXTURE:

MOLARITY VOL FINAL CONC

MgCl2	0.6M	.05 mL
NaCl	1.5M	.9	150mM
KCl	1.5	.2	33 mM
ATP	.02	1.3	2.88 mM
TRIS	1 M	1.8mL	200 mM
PROTEIN	*	1.2 mL
WATER		3.55 mL
TOTAL		9.0 mL

* TO BE DETERMINED BY BIORAD USING MATERIAL LEFTOVER

THIS GIVES YOU ENOUGH FOR FOUR 2 ML SAMPLES, A ZERO AND THREE OTHERS.

TAKE OUT SAMPLES AS FOR MYOSIN ATPASE AND DETERMINE THE Pi CONTENT AS FOR MYOSIN ATPASE. THIS ENZYME IS LESS ACTIVE, SO USE LONG TIME PERIODS LIKE 5,10,15 MINUTES AT 35 DEGREES.

SUBSTITUTE NITROPHENYLPHOSPHATE IN THE REACTION AND READ ON THE SPECTROPHOTOMETER DIRECTLY, WITHOUT THE ADDITION OF THE Pi DETECTION REAGENTS.

VARY THE ASSAY BY LEAVING OUT BOTH Na+ AND K+ TO SEE THE LEVEL OF Mg++ ATPASE. TO MAKE SURE IT IS NOT A Ca++ ATPASE, SUBSTITUTE Ca++ FOR Mg++ IN ONE EXPERIMENT, OR ADD EGTA TO THE SAME MOLARITY AS THE Mg++.

MITOSIS REGULATION

Prepare fertilized eggs as in the fertilization exercise. Resuspend .1 ml eggs into ten ml of the following drugs in seawater and pour into a petri dish; all at 10 uG/ML

1. D-ACTINOMYCIN

2. PUROMYCIN

3. COLCHICINE

4. VINBLASTINE

5. CYTOCHALASIN

6. DEXAMETHASONE

7. DNP

8. ARSENIC

9. CYANIDE

10. Place .1 ml eggs into normal seawater as a control.

11. Place a similar control on ice.

12. Place a control at 37 degrees.

Observe to see when cleavage occurs, or if it occurs. Determine whether each drug or treatment should work at the nuclear level or spindle level, or cleavage furrow level, or metabolic level.

ISOLATION OF MITOTIC APPARATUS

Remove the vitelline membrane from unfertilized eggs by treatment in 3 mM dithiothreitol (DTT), pH 9 for ten minutes. Decant off the supernatant from the settled eggs (they will settle during the ten minutes.) Wash 2x with sea water and then fertilize as in fertilization exercise. You will not be able to see a membrane elevate, since it is gone, so it would be a good idea to reserve a few eggs that are not treated with dtt to fertilize at the same time, to be sure the sperm is good. After fertilized eggs settle, resuspend them in Ca-Mg-free sea water (CMFSW) which contains 10 mM EDTA, pH 7.5. Settle, repeat. When nuclear membrane breaks down in 1 1/2 to 2 hours remove the supernate and replace with 5 volumes of 90% 1m dextrose, 10% cmfsw. When they reach metaphase, suspend the cells in 10 volumes of isolation medium, stand 2 mins, then shake moderately to break cells, or pass through 54 um nitex cloth, place in an ice bath, and observe to see that cells are broken. Spin at 200 g to sediment mitotic apparatus (at 0 degrees for 30 min.) Add CaCl2 to a concentration of .1 mM to halve the mitotic apparatus. Observe and freeze

ISOLATION MEDIUM 1 M SUCROSE, .15 M DITHIODIGLYCOL, 1 mM EDTA pH6.4. An alternative is 20 mM MES, 10 mM EGTA, 1 mM MgCl2 pH 6.4.

REFERENCES: Mazia et al. The direct isolation of the mitotic apparatus. J. Biophys biochem cytol. 10: 467-74. 1961. Silver et al. Isolation of mitotic apparatus containing vesicles with calcium sequestration activity. Cell 19:505-16, 1980.

ISOLATION OF TUBULIN

One half the class will use chick brains and one half will use sea urchin eggs as a source of tubulin. Dissection kits will be needed for chick brain preparation. From chick brains ten chicks 15-17 days old (ours are 19 days) in incubation (hatching is 21 days) should be sacrificed by decapitation and the brains cut out and weighed. ( Save the heart,liver, eyes of the chick for later lipid experiments, freeze in separate containers, well labelled.) After mincing with scissors, brains will be homogenized in 1.5 Volumes (1.5 ml x g weight) buffer

20 mM NaPO4, pH 6.75, 10mM Na glutamate, .02% sodium amide, 0.1mM ATP or 0.1M PIPES, containing 1 mM EGTA and 0.1 mM GTP pH 6.94. Centrifuge at 20,000 rpm in Sorval for 60 min. Keep the supernate, measure the volume, and adjust to 1 mm GTP. Dialyze overnight or 2 days against PIPES buffer. This is a crude prep of tubulin. Raise the temperature to 37 'C. Run a turbidometric assay. Polymerize for 30 min at 37 degrees, then run another set of assays. Now add 2 mM taxol and polymerize for another 30 min, run assays. Spin at 18,000 rpm for 30 min. Save the pellet. Resuspend in .14 the volume that was present in the original homogenate. Store in refrigerator or freeze by placing tube in dry ice and acetone and store in freezer. To use, thaw. Run proteins to determine concentration: do 1/5, 1/10, 1/50 dilution. Then to calculate concentrations for all these dilutions, figure ug/ml protein for the OD you got for each dilution, then multiply by the dilution (5,10,or 50) and divide by 1000, that equals mg protein/ ml. Make a table of these values. Reference; Borisy et al, NY Acad Sci Proc. 1974 p 107. Purification of tubulin, etc.

Store in freezer, well labelled. For polymerization, other optimal conditions are: 5mM BES ph 6.5 ; .5 mM MgSO4 ; 1 mM GTP ; 1 mM EGTA ; 50 mM KCl ; 1.6 mg/ml tubulin (final concentration, not before adding the other reagents). Stop read the next paragraphs before adding anything to reach these concentrations, add more concentrated reagents, and bring protein concentration from what it was before dilution. You need 5 ml for viscometer and you may only have enough protein for one run so be careful of the order of addition of reagents. Addition of Ca++ and colchicine can inhibit polymer formation use final concentrations of: 1.6 mM CaCl2, 10-5 M colchicine addition of deuterated water can promote polymerization. Dilute the tubulin prep with 30% the volume of deuterated water(D2O). So when designing an experiment: make your reaction mix in the cold, then run viscosity, in the warm water bath, then add Ca and run again, then try the same whole series again, but add colchicine instead of Ca++.Run viscosity measurements for at least 20 min after adding reagent. Note change in viscosity with time (hopefully). Turbidometric assay of polymerization follow optical density at 350 and 450 nm. You should get an increase as the temperature goes up. Cool to 10 degrees to get a decrease, which takes only 5 min at that temperature. You could also try the effect of redox agents on this change in OD or see if they reverse it.

ANOTHER WAY TO ISOLATE FROM BRAINS: 0.1M MES, 1mM EGTA, 1mM GTP, o.5M MgCl2 pH 6.4. homog in ice. centrifuge 100,00 g 1 hr 4 degrees, discard pellet, mix with equal vol assembly buffer below.

ANOTHER WAY TO GET POLYMERS Reference: Y Song, S Heins, E Mandelkow, E Mandelkow. Aluminum fluoride, micrtubule stability and kinesin rigor. In MotorProteins Ed. RA Cross and J Kendrick-Jones. J. of Cell Science 1991 Supplement 14. The assembly buffer from this paper is as follows:

0.1M PIPES pH 6.9, 1mM each MgSO4, EGTA, DTT, and GTP. Add NaF and AlCl3 ratio of 4:1- try 1mM AlCl3, 4mM NaF. Add final conc 8M glycerol. These MT should have attached kinesin.

TUBULIN PREPARATION FROM SEA URCHIN EGGS This is the method of Kuriyama (J. Biochem 81:1115-1125. 1977. You can use unfertilized eggs or eggs at first metaphse for the isolation. Eggs are washed 2x with 10 mM Na phosphate buffer, 10mM MgCl2, .24 M sucrose, pH 6.8. Pellet eggs, resuspend in 2-5 vol of 0.1 M Na phosphate, 1 mM EGTA, .5 mM MgSO4, 1 mM ATP, pH 6.7 and homogenize and spin 20,000g 30 min., in the cold. Do a batch adsorption onto DEAE-sephadex A50 ion exchanger by adding to 30 ml of supernate 10 ml of resin (activated in PMA), stir occasionally for 30 min. Pellet the resin with attached tubulin at 2000g for 1 min. Save this supernatant for electrophoresis, label it egg homog minus tubulin. Wash the resin in 30 ml 0.4 M NaCl-PMA by allowing it to stand for 10 min in the solution with occasional stirring. Spin (save the first wash for electrophoresis) and wash 2x more. The tubulin is still on the resin (hopefully). Now suspend the resin in 10 ml 0.6 M NaCl-PMA to elute the tubulin. Add glycerol to stabilize during dialysis overnight against polymerization solution, in the cold. Concentrate by pressure dialysis. Freeze for electrophoresis after assay.

ELECTROPHORESIS PRE-LAB

COMPONENTS:

What is the function of the

a) acrylamide

b) bis-acrylamide

c) ammonium persulfate

d) tris buffer

e) SDS

f)TEMED

What is the difference between and the purpose of the stacking gel and the separating gel?

Look through your textbook to find 2 different protein electrophoretic gels. What were they trying to demonstrate with the gel?

What is the purpose of running standards with your unknowns?

What kinds of stains can be used on gels to distinguish between sugar groups, phosphate groups, lipid groups on proteins?

How can you fix proteins in the gel after they are run?

When you look at a gel, how would you know which were small and which were large in MW?

Which end of the gel was at the anode?

How would that differ if you wanted to run basic proteins like histones?

How would you decide which %gel to use for your particular protein?

What is the difference between isoelectric focusing and SDS gel preparations?

What is a Western blot? How can that be more useful than just the gel?

When would you use a gradient gel?

When you want to use an enzyme assay to detect which band is your protein, what kind of electrophoresis would you use?

What is the advantage to 2-D gel electrophoresis?

SAFETY:

What safety measures are needed?

1. Use plastic gloves with acrylamide when pouring gels. Wear safety glasses when doing it in case it splashes while removing bubbles from under the template.

2. Measure TEMED in running hood with autopipette.

3. Use plastic gloves with CuCl2 stain.

4. Wash hands after all these manouvers.

ACRYLAMIDE SLAB GEL ELECTROPHORESIS

STOCK SOLUTIONS

ACRYLAMIDE 30% use plastic gloves to make up acrylamide solutions.

Acrylamide 30 g

bis .8 g

water up to 100ml

After stirring for 1 hr on magnetic stirrer (not so hard that you splash) filter through #1 whatman filter paper, store in refrigerator with your name and the date made.

LOWER TRIS (1.5 M TRIS-CL, PH 8.8) 18.17 G TRIS in about 80 ml water. Adjust to pH 8.8 with conc. HCl. Use pH meter with magnetic stirrer. Bring up to 100 ml in a volumetric. Store in a reagent bottle in refrigerator, mark name and date.

UPPER TRIS 0.5 M PH 6.8 6.06 g tris in about 80 ml water. Adjust as lower tris with pH meter, magnetic stirrer, conc. HCl to pH 6.8, bring to 100 ml with a volumtric, store as above.

BATH BUFFER pH 8.4 4X 12.0 g Tris 57.6 g glycine these do not have to be reagent grade. adjust pH when dissolved in about 500ml water, then bring to 1 L.

USE 160 ML PER ELECTROPHORESIS CHAMBER, DILUTE TO 600 ML. For SDS runs, add ml SDS to give a final concentration of 0.1%.

GEL OVERLAY 10 UL 10%AP, 4 ml water

10% AP .1G AMMONIUM PERSULFATE+1ML WATER, KEEP ONLY FOR TWO DAYS.

DESTAIN SOLUTION for 1 liter

INGREDIENT FINAL CONCENTRATION VOLUME

METHANOL 10% 100ml

ACETIC ACID 10% 100 ml

WATER 80% 800 ml

STAINING SOLUTION FOR 1 L

ISOPROPYL ALCOHOL 25% 250 ML

ACETIC ACID 10% 100 ml

0.1% COOMASSIE BLUE 1 g

WATER 65% 650 ml

GEL PREPARATION for isozyme gels

10% SEPARATING (LOWER) GEL

FOR THICK PLATES 1		2		FOR THIN PLATES 1
LOWER TRIS	5.5ML		11.0	4.5
ACRYLAMIDE	7.3		14.6	6
WATER	9.2		18.3	7.5
10%AP	100UL		200UL	50 UL
TEMED	10 UL		20 ul	10 ul
total	22.11		44.12	18.06

do not pipette acrylamide by mouth, IT IS A NEUROTOXIN, leave ingredients at room temperature until they warm up, before using. Add TEMED in fume hood, IT IS CARCINOGENIC. If you get any acrylamide on you, wash your skin, as it can be absorbed through the skin.

STACKING GEL 4.5% gel
	thin	thick
UPPER TRIS	1.5	2.5
ACRYLAMIDE	.9	1.5
10%AP	30ul	50ul
WATER	3.6ml	6 ml
TEMED	10ul	20ul

allow gels to polymerize undisturbed. Leave some in the pipette to see when it polymerizes. Do not disturb the pipette for at least 15 min, as pipetting prevents polymer formation.

SAMPLE PREPARATION FOR NATIVE ISOZYME GELS Homogenize eggs or chick embryo parts in 0.3M NaCl, tris pH 8; or in .3M sucrose, tris pH8. Weigh the tissue and use 4x dilution.

Add 10 ul tracking dye, 5 crystals of sucrose per .2 ml sample. Use 60 ug of a mixture of proteins, use 4 ug of a pure one.

Run gels for isozyme study at 10 ma/gel overnight until approximately 3 or 4 hours after the tracking dye goes off the end.

G6PD ENZYME ASSAY ON GELS AFTER THEY ARE RUN.

REACTION MIXTURE

A NADP 1MG/.4 ML 10X TEA- MAKE FRESH EVERY DAY

B KCN 13.2 MG/2ML WATER (MULTIPLY AMOUNT BY NUMBERS OF GELS RUN

C NITRO-BLUE TETRAZOLIUM 4.9 MG/2 ML WATER

D PHENAZINE METHOSULFATE 2.14 MG/10ML WATER

KEEP B,C,D IN DARK BOTTLES make up ABCD EVERY DAY FRESH

E G6P (ADJUST TO pH 7.4 (10x kept in freezer)

TEA BUFFER 10X KEPT IN FREEZER

.05M TRIETHANOLAMINE 7.46G

.01M MgCl2 .952 g

.005 M EDTA 1.46G

ADJUST TO pH 7.4 and then bring to 100 ml and freeze in small aliquots

After stopping current, remove gels by separating plates with spatula. BE SURE TO MARK YOUR GEL AT SLOT NUMBER 1 WITH CUTTING OFF THE CORNER DIAGONALLY. Cut off the stacking gel just above the top of the separating gel. Drop into 20 ml 1 x TEA and place on shaker for 10 min.

Prepare reaction mixture as follows:

NADP	0.16 ML
KCN	2 ML
NITROBLUE	2 ML
PHENAZINE	2 ML
G6P	2 ML
BUFFER	2 ML
WATER	10 ML
FINAL VOLUME	20 ML

Pour off TEA, holding gel in plate with rubber gloves (finger marks will appear on gel otherwise. Place gel on slowly moving shaker (in a covered box, must be in the dark) for about 2 hr. Rinse 5-6x with distilled water. Shake gently with enough 10% methanol, 1% glycerol to cover for at least 4 hr, then mount for drying under vacuum.

MEASUREMENT OF MOLECULAR WEIGHT USING PAGE ISOZYME SEPARATIONS REFERENCE:J.L. Hedrick, A.J. Smith. 1968. Arch. Biochem. Biophys. 126:155-64. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis.

Purpose: to distinguish between polymer isozymes differing mainly in size and distinct isozyme proteins differing in size and charge.

1. Calculate the relative mobility of the isozyme band or standard protein stained band relative to the tracking dye for gels of different % acrylamide concentration.

2. Plot log Rm (relative mobility) versus % gel on graph paper or plot relative mobility vs % gel on semilog paper. Calculate the slope and compare for each protein standard and isozyme. Lines which are non-parallel and converge on one y intercept are polymers of one subunit. Proteins with similar size, but different charge have parallel lines with the same slope. Proteins differing in both molecular size and charge will have non-parallel lines which do not intersect the y axis together. 3. Calculate the molecular weight of the unknowns by reading it off of a plot of standard molecular weight proteins vs slope.

ESTIMATION OF SUBSTRATE SPECIFICITY

Record which isozymes are present in various substrates and cofactors, and their relative intensities . Draw some conclusions about specificity from your data.

LACTIC DEHYDROGENASE ISOZYMES

Comparison of isozymes can be made from different tissues, especially heart and skeletal muscle, or from different stages of embryonic development. Different tissues have different tolerances for high levels of lactic acid buildup and their enzymes are different.

Homogenize the tissue with sucrose homogenizing medium. Run the electrophoresis as for G6PD. Make sure that the electrophoresis was allowed to run for about 4 hours after