Evonik_Agarose Gels for Proteins

8/2/2019 Evonik_Agarose Gels for Proteins

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EVT 011285AM

The Biotechnology Education Company

The Biotechnology Education Company 1-800-EDVOTEK www.edvotek.com

All components are intended for educational research only.

They are not to be used for diagnostic or drug purposes, nor

administered to or consumed by humans or animals.

111EDVO-Kit #

Electrophoretic Propertiesof Native Proteins

Storage:

See page 3 for specific

storage instructions.

EXPERIMENT OBJECTIVES:

The objective of this experiment is todevelop a general understanding of the

structure and electrophoretic migrationof native proteins.


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EDVO-Kit # 111 Electrophoretic Properties of Native Proteins

EVT 011285AM

Table of Contents

Page

Experiment Components 3

Experiment Requirements 3

Background Information

Electrophoretic Properties of Native Proteins 4

Experiment Procedures

Experiment Overview 7

Preparations for Agarose Gel Electrophoresis 8

Practice Gel Loading 11

Conducting Agarose Gel Electrophoresis 12

Staining the Gel 13

Study Questions 14

Instructor's Guidelines

Notes to the Instructor 15

Pre-Lab Preparations 16

Quick Reference Tables 17

Avoiding Common Pitfalls 18

Idealized Schematic of Results 19

Study Questions and Answers 20

Material Safety Data Sheets 22


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EDVOTEK - The Biotechnology Education Company

1-800-EDVOTEK www.edvotek.com24-hour FAX: (301) 340-0582 email: [email protected]


EVT 011285AM

A Bovine Serum Albumin (BSA)

B OvalbuminC Cytochrome C

D LysozymeE Horse Serum Proteins

Practice Gel Loading Solution

UltraSpec-Agarose Powder

50x Electrophoresis Buffer Protein InstaStain Sheets

1ml Pipet 100ml Graduated Cylinder

(packaging for samples)

Microtipped Transfer Pipets

Experiment Components

Horizontal Gel Electrophoresis Apparatus

D.C. Power Supply

Automatic Micropipets with Tips Waterbath

Recommended Equipment:Visualization System (white light)

Glass Staining Tray 250ml Flasks Pipet Pump

Hot Gloves Marking Pens

Distilled or Deionized Water Methanol

Glacial Acetic Acid

Requirements

All components are

intended foreducational research

only. They are not tobe used for

diagnostic or drug

purposes, nor

administered to orconsumed byhumans or animals.

UltraSpec-Agaroseand Protein Plus are

trademarks ofEDVOTEK, Inc.

Protein InstaStain,

EDVOTEK, and The

BiotechnologyEducation Company

are registered

trademarks ofEDVOTEK, Inc.

Store ComponentsA-E at 4C.

This experimentcontains ready-to-load

protein samples andreagents sufficient for

6 gels (see QuickReference).

Quick Reference:

There is enough sample for6 gels if you are using anautomatic micropipet for sampledelivery. Use of transfer pipetswill yield fewer gels.


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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purposewithout the written consent of EDVOTEK, Inc. Copyright 1989,1992,1994,1995,1997, 1998, 1999, 2000, 2005EDVOTEK, Inc., all rights reserved. EVT 011285AM

BackgroundInformation


Properties of Native Proteins

Proteins are a highly diversified class of biomolecules. Differences in theirchemical properties, such as charge, shape, size and solubility, enable

them to perform many biological functions. These functions include

enzyme catalysis, metabolic regulation, binding and transport of smallmolecules, gene regulation, immunological defense and cell structure.

A protein can have a net negative or positive charge, depending on its

amino acid composition and pH conditions. At a certain pH, the mol-

ecule can be electrically neutral, i.e. negative and positive charges areequal. In this case, the protein is isoelectric. In the presence of an

electrical field, a protein with a net negative or positive charge willmigrate towards the electrode of opposite charge. The amino acid

residues responsible for a proteins negative charge at physiological pH

are glutamic acid and aspartic acid. A proteins positive charge atphysiological pH is due to lysine, arginine and, to a lesser extent, histidine.

Figure 1:Effect of pH on Peptide Containing Glutamic and Lysine Residues

H|

|H

CH2- CH

2- CH

2- CH

2- N - H

CO

HO

CH2- CH

2- C

H|

H-N-H

+

+

OH

O

Acidic pH

( < 4 )

CH2- CH

2- CH

2- CH

2- N - H

CO

O

CH2- CH

2- C

H|

H-N-H

+

+H|

|H

CH2- CH

2- CH

2- CH

2- N

CO

O

CH2- CH

2- C

H|

H-N-H

+

H|

|H

OH

O

OH

O

- -

Alkaline pH

( > 9 )

Neutral pH

( 7 )

N-Terminal

GlutamicAcid

Lysine

C-Terminal

At acidic pH, glutamic acid and aspartic acid residues have little charge,while lysine and arginine both have positive charges. As the pH of the

protein solution is raised, glutamic and aspartic acid release a proton andbecome negatively charged. However, lysine and arginine residues

become uncharged as the pH is raised to high values. (See Figure 1.)

The direction and extent of a proteins migration in an electric field can

be altered by using an acidic, neutral or alkaline buffer system duringelectrophoresis. The isoelectric point of a protein is defined as the pH at

which the protein has no net charge. Consequently, a protein will notmigrate in an electric field at its (pI) isoelectric point. An isoelectric

protein still possesses areas of negative and positive charge, but overall


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5

BackgroundInfo

rmation



they cancel each other. Proteins that contain more aspartic andglutamic acid residues will have isoelectric points at acidic pH values.

Conversely, proteins that contain more lysine and arginine residues will

have isoelectric points at alkaline pH values.

Some proteins contain other chemical groups in addition to their aminoacid residues. For example, hemoglobin and cytochrome c contain

heme, which consists of iron complexed to a system of organic rings

called porphyrin. Non-amino acid structural groups covalently at-tached or very tightly bound to the polypeptide chain(s) of a protein are

called prosthetic groups. Prosthetic groups enable proteins to performbiological functions and have a strong influence on the proteins chemi-

cal properties. The oxygen atoms transported by hemoglobin are actu-

ally bound at the heme irons.

Proteins exhibit many different three-dimensional shapes and complexfolding patterns which are determined by their amino acid sequence and

post translational processing such as adding carbohydrate residues orprosthetic groups. The precise three-dimensional configuration of a

protein is critical to its biological function. The general shapes for proteins

are spherical, elliptical or rod-like. The molecular weight is a function ofthe number and type of amino acids in the polypeptide chain. Proteins

can consist of a single polypeptide or several polypeptides specificallyassociated with each other. These polypeptides can be identical, similar

or completely different from one another. The number and nature of

polypeptides in a protein has large effects on its mass, size and shape.Proteins that are in their normal, biologically active forms are called

native. Certain detergents, extremes of pH, organic solvents and heatcan cause a native protein to lose its specific three-dimensional folding

pattern and, consequently, its biological activity. Proteins that have gonethrough this process are called denatured. Denatured proteins can haveradically different behavior from their native forms during electrophoresis.

ELECTROPHORESIS OF PROTEINS

The properties of proteins affect the way they migrate during gel electro-

phoresis. Gels used in electrophoresis (e.g. agarose, polyacrylamide)consist of microscopic pores of a defined size range that act as a molecu-

lar sieve. Only molecules with net charge will migrate through the gel

when it is in an electric field. Small molecules pass through the poresmore easily than large ones. Molecules having more charge than others

of the same shape and size will migrate faster. Molecules of the samemass and charge can have different shapes. In this case, those with a

more compact shape, like a sphere, will migrate through the gel morerapidly than those with an elongated shape, like a rod. In summary, the

amount and sign of charge, the size and shape of a native protein, all

affect its electrophoretic migration rates.


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BackgroundInformation



Electrophoresis of native proteins is useful in clinical and immunologicalanalysis of complex biological samples, such as serum. Serum consists of

many different types of proteins. Gel electrophoresis of native serum

proteins at alkaline pH results in several zones. Albumin is by far the mostabundant serum protein and has one of the fastest electrophoretic

migration rates. Albumin binds and transports many small molecules,including fatty acids and bilirubin. It is also involved with osmotic regula-

tion. The serum proteins with the slowest migration rates are the gamma

globulins (antibodies). In between these two zones several other types ofproteins can be observed. These include transferrin (iron transport),

ceruloplasmin (copper transport), macroglobulin (protease inhibitor) andhaptoglobin (involved with the binding and conservation of hemoglobin).

Electrophoretic patterns of human serum proteins can aid in the diagnosis

of certain diseases. For instance, cirrhosis of the liver causes a decrease inalbumin, while multiple myeloma ( a cancer of the immune system) and

chronic rheumatoid arthritis causes abnormal increases in the gammaglobulins.

The purified proteins used in this experiment all consist of single polypep-

tide chains, but differ in their charge and mass. Bovine serum albumin,

with a molecular weight of 68,000 and an isoelectric point (pI) of 4.7, issimilar in structure and function to the serum albumin previously discussed.

Ovalbumin is an abundant protein found in eggs. Ovalbumin, with amolecular weight of 43,000 and pI of 4.6, contains covalently attached

carbohydrate and is therefore a glycoprotein. The polysaccharide chain

contains eight (8) sugar residues. The majority of serum proteins are alsoglycoproteins (except serum albumin). Cytochrome C, a ubiquitous

protein with a molecular weight of 12,000 and pI of 10.7, is involved inelectron transport reactions that are mediated by the heme prosthetic

group. Cytochrome C and several other proteins form the electron-transport chain which enables the cell to obtain chemical energy (ATP)from the oxidation of glucose. In bacteria, cytochrome c is located on

the inner surface of the cellular membrane. Lysozyme, a small proteinwith a molecular weight of 14,000 and pI of11.2, is utilized in bacterial cell

lysis. All these proteins are dissolved in a buffer containing glycerol andthe negatively charged bromophenol blue tracking dye. This tracking

dye generally migrates faster than the proteins. During the electrophoresis

of the serum proteins, the bromophenol blue may separate into twobands. This separation occurs because some of the serum proteins bind a

fraction of the dye. The bound dye has a slower migration rate than thefree dye.

After electrophoresis, the proteins will be visualized by staining with Protein

InstaStain After the gel is destained, proteins will appear as dark bluezones against a light blue background.


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ExperimentProc

edures


Wear gloves

and safetygoggles

Experiment Overview

EXPERIMENT OBJECTIVE:

The objective of this experiment module is to develop a general under-

standing of the structure and electrophoretic migration of native proteins.

LABORATORY SAFETY

1. Gloves and goggles should be worn routinely as

good laboratory practice.

2. Exercise extreme caution when working with equip-ment which is used in conjunction with the heating

and/or melting of reagents.

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS

OR BULBS.


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ExperimentProcedures

Wear gloves

and safety goggles

Preparations for Agarose Gel Electrophoresis

PREPARING THE GEL BED

1. Make sure the gel bed is clean and dry.

2. Close off the open ends of the bed by usingrubber dams or tape.

A. Using Rubber dams:

Place a rubber dam on each end of the bed.

Make sure the rubber dam sits firmly incontact with the sides and bottom of the bed.

B. Taping with labeling or masking tape:

With 3/4 inch wide tape, extend the tape over the sides and

bottom edge of the bed.

Fold extended edges of the tape back onto the sides andbottom. Press contact points firmly to form a good

seal.

3. Place the well forming template (comb)

across the bed in the middle set of

notches. The comb should sit firmly andevenly across the bed.

CASTING THE AGAROSE GEL

This experiment requires a 0.8% gel.

3. Use a 250ml flask to prepare the diluted gel buffer.

With a 1ml pipet, measure the buffer concentrate and add

the distilled water as indicated in Table A.

4. Add the required amount of agarose powder. Swirl to disperseclumps.

5. With a marking pen, indicate the level of the solution volume onthe outside of the flask.

Amt of

Agarose

(g)

Concentrated

Buffer (50x)

(ml)

Size of EDVOTEK

Casting Tray

(cm)

Distilled

Water

(ml)

Total

Volume

(ml)

7 x 15 0.48 1.2 58.8 60

+ =+

Table A Individual 0.8% UltraSpec-Agarose Gel


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9

ExperimentProc

edures


6. Heat the mixture to dissolve the agarose powder. The final solution

should be clear (like water) without any undissolved particles.

A. Microwave method: Cover flask with plastic wrap to minimize evaporation.

Heat the mixture on high for 1 minute. Swirl the mixture and heat on high in bursts of 25 seconds

until all the agarose is completely dissolved.

B. Hot plate or burner method:

Cover the flask with foil to prevent excess evaporation. Heat the mixture to boiling over a burner with occasional

swirling. Boil until all the agarose is completely dissolved.

7. Cool the agarose solution to 60C with careful swirling to

promote even dissipation of heat. If detectable evapo-ration has occurred, add distilled water to bring the

solution up to the original volume as marked on the flask

in step 5.


After the gel is cooled to 60C:

If using rubber dams, go to step 9. If using tape, continue

with step 8.

8. Seal the interface of the gel bed and tape to prevent

the agarose solution from leaking. Use a transfer pipet to deposit a small amount of

cooled agarose to both inside ends of the bed. Wait approximately 1 minute for the agarose tosolidify.

9. Pour the cooled agarose solution into the bed. Make

sure the bed is on a level surface.

10. Allow the gel to completely solidify. It will

become firm and cool to the touch afterapproximately 20 minutes.

60C

DO NOT POUR BOILING HOTAGAROSE INTO THE GEL BED.

Hot agarose solution mayirreversibly warp the bed.

Caution!


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PREPARING THE SOLIDIFIED GEL FORELECTROPHORESIS

11. Carefully remove the rubber dams or tape.

12. Remove the comb by slowly pulling it straightup. Do this carefully and evenly to prevent

tearing the sample wells.

13. Inspect the wells by viewing the gel from the

edge nearest the wells. If some of the wellsare ripped through their bottoms or sides, do

not use them when loading samples.

14. Place the gel in the electrophoresis chamber,properly oriented, centered and level on the platform.

15. Fill the chamber of the electrophoresis apparatus with the required

volume of diluted buffer as outlined in Table B.

16. Load samples in wells; conduct electrophoresis according to experi-ment instructions. See Table C for time and voltage guidelines.

Step 11: Be careful notto damage or tear thegel when removing rubberdams. A thin plastic knife orspatula can be inserted betweenthe gel and the dams to breakpossible surface tension.

Useful Hint!

ConcentratedBuffer (50x)

(ml)

EDVOTEK

Model #

Distilled

Water(ml)

Total

Volume(ml)

Table B Electrophoresis Buffer

M6+

M12

M36 (blue)

M36 (clear)

6

8

10

20

294

392

490

980

300

400

500

1000

=+


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ExperimentProc

edures


Quick Reference:

If you are using an automaticmicropipet, deliver 20 microlitersto the sample well. If usingtransfer pipets, load the samplewell until it is full.

Use microbiology-grade agar andwater to make practice gels - savethe agarose for the experiment.

Practice Gel Loading

EDVOTEK experiments which involveelectrophoresis contain practice gel loading

solution. If your students are unfamiliar with

loading samples in agarose gels, it issuggested that they practice sample

delivery techniques before performingthe electrophoresis part of an experi-

ment. Using the EDVOTEK system, sampledelivery can be performed by using either

an automatic micropipet, or dispos-

able microtipped transfer pipets.

Casting of a separate practice gel ishighly recommended. One suggested

activity for practice gel loading is outlined

below:

1. Cast a gel with the maximum number of wells and placeit under the buffer in an electrophoresis apparatus cham-

ber. (Use microbiology-grade agar and water to make

practice gels - save the agarose for the experiment.)

2. Let students practice delivering the practice gel loadingsolution to the sample wells.

3. If students need more practice, remove the practice gel

loading solution by squirting buffer into the wells with a

transfer pipet.

4. When students are finished practicing, replace the prac-tice gel with a fresh gel and continue with the experiment.

The practice gel loading solution will become diluted inthe buffer and will not interfere with the experiment.

Remember!


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Electro

phoresi

s

M12

Conducting Agarose Gel Electrophoresis

Reminder:

During electrophoresis, the DNAsamples migrate through theagarose gel towards the positiveelectrode. Before loading thesamples, make sure the gel isproperly oriented in the apparatuschamber.

This experiment requires a 0.8% UltraSpec-Agarose gel.The gel should be cast with the comb placed in the middle

set of notches of the gel bed. Make sure the electrophoresisapparatus leads reach the power source before loading

samples. The apparatus should not be moved after the

samples are loaded because movement of the unit willcause samples to spill out of the wells.

LOADING PROTEIN SAMPLES

1. Consecutively load 40l of each sample in

tubes A - E into wells in the middle of thegel.

RUNNING THE GEL

2. After the samples are loaded,

carefully snap the cover down

onto the electrode terminals.

Make sure that the negative

and positive indicators onthe cover and apparatus

chamber are properlyoriented.

3. Insert the plug of the black

wire into the black input of

the power source (negativeinput). Insert the plug of the

red wire into the red input ofthe power source (positive

input).

4. Set the power source at the required voltage and run

the electrophoresis for the length of time as determinedby your instructor. When current is flowing properly, you

should see bubbles forming on the electrodes.

5. After the electrophoresis is completed, turn off the

power, unplug the power source, disconnect the leadsand remove the cover.

VoltsRecommended Time

Minimum Optimal

125 30 min 45 min

70 40 min 1.5 hrs

50 60 min 2.0 hrs

Time andVoltage

Table C:

+-Black Red

Sample wells

Useful Hint!

Afterelectrophoresis,remove a small slice ofthe gel from the upper

right hand corner to easilyidentify right and leftorientation of the gel afterstaining.


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ExperimentProc

edures


ONE-STEP STAINING & DESTAINING WITH PROTEININSTASTAIN

Protein agarose gels can be stained with Protein InstaStain cards in oneeasy step an excellent alternative if time does not permit staining

during a regular class session.

1. After electrophoresis, submerge the gel and plate in a small tray with

100ml of fixative solution. (Use enough solution to cover the gel.)

2. Gently float a sheet of Protein InstaStain with the stain side (blue) in

the liquid. Cover the gel to prevent evaporation.

3. Gently agitate on a rocking platform for 1-3 hours or overnight.

4. After staining, protein bands will appear as dark blue bands againsta light background and will be ready for photography.

NO DESTAINING IS REQUIRED.

5. If the gel is too dark, destain in several changes of fresh destain

solution until the appearance and contrast of the protein bandsagainst the background improves.

Staining the Gel

Fixative and DestainingSolution for each gel

(100ml)

50ml Methanol10ml Glacial Acetic Acid40ml Distilled Water


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1. Under the conditions of the electrophoresis (pH 7.8), which of theproteins (BSA, ovalbumin, Cytochrome C, Lysozyme) are net positive?

Which are net negative?

2. Based on your observations, would you expect ovalbumin to have

more or less lysine and arginine residues than lysozyme? Why?

3. A sample of native protein is submitted to native gel electrophoresis.After the electrophoresis was run for several hours, it was found that

the protein did not enter the gel from the sample well (did not

migrate). What is the most likely explanation for this observation?What experimental condition could you change that would allow the

protein to migrate?

4. Consider the following information about the proteins used in this

experiment:Approximate Isoelectric

Protein Molecular Weight Point (pH)Cytochrome C 12,000 10.7

Lysozyme 14,000 11.2Ovalbumin 43,000 4.6

BSA 68,000 4.7

All these proteins are spherical in shape, therefore, increasing mo-

lecular weight corresponds to increasing size. The results of the

electrophoresis at pH 7.8 should show that ovalbumin migrates agreater distance towards the positive electrode than BSA. The results

should also show that Lysozyme migrates a greater distance towardsthe negative electrode than Cytochrome C. Explain the results using

the information provided above.

Study Questions


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Instructor'sGuide


15EDVO-Kit # 111 Electrophoretic Properties of Native Proteins

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Notes to the Instructor

APPROXIMATE TIME REQUIREMENTS FOR

PRE-LAB AND EXPERIMENTAL PROCEDURES

1. Agarose gel preparation: Your schedule will determine when to

prepare the agarose gel(s). Whether you choose to prepare thegel(s), or have the students do it, allow approximately 30 to 40 minutes

for this procedure. Generally, 20 minutes of this time is required for

gel solidification.

2. The approximate time for electrophoresis will vary from 45 minutes to 2hours.

A variety of factors, such as class size, lengthof laboratory sessions, and availability of

equipment, will influence the implementationof this experiment with your students. These

guidelines can be adapted to fit your specific

set of circumstances.

SPECIFIC REQUIREMENTS FOR THISEXPERIMENT

Gel Concentration

This experiment requires a 0.8% UltraSpec-Agarose gel.

Number of Wells Required

This experiment requires a gel with 5

sample wells.

Placement of Comb

During gel casting, the comb should beplaced in the notches in the middle of the

gel bed.


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Instructor'sGuide

16



PreLab Preparations

The same 50xconcentrated buffer isused for preparing theagarose gel buffer andthe chamber buffer.

Useful Hint!

PREPARING THE ELECTROPHORESIS (CHAMBER) BUFFER

Prepare the appropriate volume of diluted electrophoresis chamber

buffer by mixing the concentrated 50x electrophoresis buffer and distilledor deionized water according to Table B .

ELECTROPHORESIS TIME AND VOLTAGE

Your schedule will dictate the length of time samples will be separated byelectrophoresis. General guidelines are presented in Table C.

PREPARING STAINING AND DESTAINING SOLUTIONS

Solution for staining with Protein InstaStain

Prepare a stock solution of Methanol and Glacial Acetic Acid by

combining 180ml Methanol, 140ml Distilled water, and 40 mlGlacial Acetic Acid.

No destaining is required.

Wear gloves

and safety goggles


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Instructor'sGuide



Quick Reference Tables

Amt of

Agarose

(g)


(ml)

Size of EDVOTEK

Casting Tray

(cm)

Distilled

Water

(ml)

Total

Volume(ml)

7 x 15 0.48 1.2 58.8 60

+ =+

Table A Individual 0.8% UltraSpec-Agarose Gel


(ml)

EDVOTEK

Model #

Distilled

Water

(ml)

Total

Volume(ml)

Table B Electrophoresis Buffer

M6+

M12

M36 (blue)

M36 (clear)

6

8

10

20

294

392

490

980

300

400

500

1000

=+

Table CTime and

Voltage

Recommended Time

Minimum MaximumVolts

125

70

50

30 min

40 min

60 min

45 min

1.5 hrs

2.0 hrs

Electrophoresis of DNA


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Instructor'sGuide

18



Avoiding Common Pitfalls

To ensure that protein bands are well resolved, make sure the gelformulation is correct (see Table A on page 17) and that electro-phoresis is conducted for the optimum recommended amount of

time.

Correctly dilute the concentrated buffer for preparation of the gel

and electrophoresis buffer. Remember that without buffer in the gel,there will be no protein mobility. Use only distilled water to prepare

buffers. Use distilled or deionized water.

For optimal results, use fresh electrophoresis buffer prepared accord-ing to instructions.

Before performing the actual experiment, practice sample deliverytechniques to avoid diluting the sample with buffer during gel load-

ing.

To avoid loss of protein bands into the buffer, make sure the gel is

properly oriented so the samples are not moving in the wrong direc-tion and off the gel.

If protein bands appear faint after staining and destaining, repeat the

staining procedure but stain for a longer period of time.


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Instructor'sGuide



1 2 3 4 5 6

( + )

( - )

Idealized Schematic of Results

The figure to the left is an idealized schematic showingrelative positions of protein polypeptides. The idealized

schematic (left) shows the relative positions of the bands,but are not depicted to scale. Actual results are shown

below.

Lane Tube

1 A Bovine Serum Albumin (BSA)2 B Ovalbumin

3 C Cytochrome C

4 D Lysozyme5 E Horse Serum Proteins

1 2 3 4 5 6

( + )

( - )


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Instructor'sGuide

20



Study Questions and Answers

1. Under the conditions of the electrophoresis (pH 7.8), which of the

proteins (BSA, ovalbumin, Cytochrome C, Lysozyme) are net positive?

Which are net negative?

At pH 7.8 cytochrome c (pI 10.7) and lysozyme (pI 11.2) have a netpositive charge and therefore travel toward the negative electrode.

BSA (pI 4.7) and ovalbumin (pI 4.6) are negative and therefore travelto the positive electrode.

2. Based on your observations, would you expect ovalbumin to havemore or less lysine and arginine residues than lysozyme? Why?

Ovalbumin has less Lysine and Arginine residues and therefore is less

positive (more negative) and thus moves toward the positive elec-

trode (anode).

3. A sample of native protein is submitted to native gel electrophoresis.After the electrophoresis was run for several hours, it was found that

the protein never entered the gel from the sample well, i.e. it did not

migrate. What is the most likely explanation for this observation?What experimental condition could you change that would allow the

protein to migrate?

The buffer used for separation is pH 7.8. Assume that the proteins arespherical in shape and therefore conformation will not be a factor,

and the molecular weight will correspond directly to the size. Ovalbu-

min has a molecular weight of 43,000 and a pI of 4.6. Therefore at pH7.8, it will be negative. BSA has larger molecular weight 68,000 with

approximately the same pI of 4.7. Therefore both ovalbumin and BSA

have a net negative charge, but since BSA is larger it will move slower.

4. Consider the following information on the proteins used in this experi-ment:

Approximate IsoelectricProtein Molecular Weight Point (pH)

Cytochrome C 12,000 10.7

Lysozyme 14,000 11.2Ovalbumin 43,000 4.6

BSA 68,000 4.7

All these proteins are spherical in shape, therefore, increasing mo-

lecular weight corresponds to increasing size. The results of theelectrophoresis at pH 7.8 should show that Ovalbumin migrates a

greater distance towards the positive electrode than BSA. The resultsshould also show that Lysozyme migrates a greater distance towards

the negative electrode than Cytochrome C. Explain the results usingthe information provided above. (Answer on next page.)


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Instructor'sGuide



Study Questions and Answers

The following are possible explanations:

A. The agarose gel pores are not large enough. Therefore, protein

samples cannot penetrate the gel.

B. The protein has a net charge of zero at pH 7.8, the pH of theelectrophoresis buffer.

Possible remedies: Decrease the gel concentration to make larger pores.

Change the pH of the electrophoresis buffer.


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Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.

IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is notapplicable, or no information is available, the space mustbe marked to indicate that.

Section IManufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

Address (Number, Street, City, State, Zip Code)

EDVOTEK, Inc.

14676 Rothgeb Drive

Rockville, MD 20850

Hazardous Components [SpecificChemical Identity; Common Name(s)] OSHA PEL ACGIH TLV

Other LimitsRecommended % (Optional)

(301) 251-5990

(301) 251-5990

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1)2

Agarose

09-15-2002

This product contains no hazardous materials as defined by the OSHA Hazard Communication

Standard.

CAS #9012-36-6

For 1% solution 194 F

No data

No data

No data

No data

No data

Insoluble - cold

White powder, no odor

N.D. = No data

No data N.D. N.D.

Water spray, dry chemical, carbon dioxide, halon or standard foam

Possible fire hazard when exposed to heat or flame

None

Stability

Section V - Reactivity Data

Unstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion?Skin?

Other

Stable

HazardousPolymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

Section VII - Precautions for Safe Handling and Use

Steps to be Taken in case Material is Released for Spilled

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)Gen. dilution ventilation

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

Yes Splash proof goggles

Impervious clothing to prevent skin contact

None

X None

No data available

X None

Yes Yes Yes

Inhalation: No data available Ingestion: Large amounts may cause diarrhea

No data available

No data available

Treat symptomatically and supportively

Sweep up and place in suitable container for disposal

Normal solid waste disposal

None

None

Chemical cartridge respirator with full facepiece.




Section I

Manufacturer's Name




Date Prepared



EDVOTEK, Inc.

14676 Rothgeb Drive

Rockville, MD 20850



(301) 251-5990

(301) 251-5990

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point

Evaporation Rate

(Butyl Acetate = 1)


50x Electrophoresis Buffer

This product contains no hazardous materials as defined by the OSHA Hazard

Communication Standard.

No data

No data

No data

No data

No data

No data

Appreciable, (greater than 10%)

Clear, liquid, slight vinegar odor

No data

N.D. = No data

N.D. N.D.

Use extinguishing media appropriate for surrounding fire.

Wear protective equipment and SCBA with full facepiece

operated in positive pressure mode.

None identified

09-15-02

Stability


Unstable


Incompatibility

Conditions to Avoid


Other

Stable

Hazardous

PolymerizationMay Occur Conditions to Avoid

Will Not Occur










Other Precautions



Mechanical (General)


Protective Gloves



Eye Protection


X None

Strong oxidizing agents

Carbon monoxide, Carbon dioxide

X None

Yes Yes Yes

None

None identified

Irritation to upper respiratory tract, skin, eyes

None

Ingestion: If conscious, give large amounts of water

Eyes: Flush with water Inhalation: Move to fresh air Skin: Wash with soap and water

Wear suitable protective clothing. Mop up spill

and rinse with water, or collect in absorptive material and dispose of the absorptive material.

Dispose in accordance with all applicable federal, state, and local

enviromental regulations.

Avoid eye and skin contact.

None

Yes None

Yes None

Yes Safety goggles

None

None


23/24




Section I

Manufacturer's Name




Date Prepared



EDVOTEK, Inc.

14676 Rothgeb Drive

Rockville, MD 20850



(301) 251-5990

(301) 251-5990

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point

Evaporation Rate

(Butyl Acetate = 1)


Practice Gel Loading Solution

09-19-2002

This product contains no hazardous materials as defined by the OSHA Hazard Communication

Standard.

No data

No data

No data

No data

No data

No data

Soluble

Blue liquid, no odor

No dataNo data No data

Dry chemical, carbon dioxide, water spray or foam

Use agents suitable for type of surrounding fire. Keep upwind, avoid

breathing hazardous sulfur oxides and bromides. Wear SCBA.

Unknown

Stability


Unstable


Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur










Other Precautions





Protective Gloves



Eye Protection


X None

None

Sulfur oxides, and bromides

X None

Yes Yes Yes

Acute eye contact: May cause irritation. No data available for

other routes.

No data available

May cause skin or eye irritation

None reported

Treat symptomatically and supportively. Rinse contacted area

with copious amounts of water.

Wear eye and skin protection and mop spill area. Rinse with water.

Observe all federal, state, and local regulations.

Avoid eye and skin contact.

None

Yes None

Yes None

Yes Splash proof goggles

None required

Avoid eye and skin contact


Standard. 29 CFR 1910.1200 Standard must be consulted for

specific requirements.


Section I

Manufacturer's Name




Date Prepared



EDVOTEK, Inc.

14676 Rothgeb DriveRockville, MD 20850



(301) 251-5990

(301) 251-5990

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Protein Plus Stain

09-19-2002

Methanol (Methyl Alcohol) 200ppm 200ppm No data 90%-100%CH3OH

65C

96mmHg

1.11

.79

N/A

4.6

Complete (100%)

Blue liquid/alcoholic, pungent odor

(closed cup) 12C 6.0% 36%

Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective)

Wear SCBA with full facepiece operated in positive pressure mode.Move containers from firearea

Vapors may flow along surfaces to distant ignition sources.

Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire.

Stability


Unstable


Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur










Other Precautions





Protective Gloves



Eye Protection


X None


Carbon monoxide, Carbon dioxide, Sulfur oxides

X None

Rubber boots

Avoid prolonged or repeated exposure

Yes Yes YesIrritating to eyes, skin, mucous membranes and upper respiratory tract.

Chronic exposure may cause lung damage or pulmonary sensitization

No data No data No data

Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache

No data

Flush skin/eyes w/ large amounts of water. If inhaled, remove to fresh air. Ingestion: give large amounts

of water or milk. Do not induce vomiting.

Evacuate area. Wear SCBA, rubber boots and rubber gloves. Mop up w/ absorptive material and burn in

chemical incinerato equipped w/ an afterburner and scrubber.

Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalation.

Strong sensitizer

NIOSH/MSHA approved respirator

No Chem fume hood

No None

Rubber Splash-proof goggles


24/24



IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is notapplicable, or no information is available, t he space mustbe marked to indicate that.

Section IManufacturer's Name




Date Prepared



EDVOTEK, Inc.

14676 Rothgeb Drive

Rockville, MD 20850



(301) 251-5990

(301) 251-5990

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Protein InstaStain

09-19-2002

Methanol (Methyl Alcohol) 200ppm 200ppm No data 90%-100%CH3OH

65C

96mmHg

1.11

.79

N/A

4.6

Complete (100%)

chemical bound to paper, no odor

(closed cup) 12C 6.0% 36%

Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective)

Wear SCBA with full facepiece operated in positive pressure mode.Move containers from firearea

Vapors may flow along surfaces to distant ignition sources.

Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire.

Stability


Unstable


Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur


Carcinogenicity: NTP? OSHA RegulationIARC Monographs?








Other Precautions





Protective Gloves



Eye Protection


X None


Carbon monoxide, Carbon dioxide, Sulfur oxides

X None

Rubber boots

Avoid prolonged or repeated exposure

Yes Yes YesIrritating to eyes, skin, mucous membranes and upper respiratory

Chronic exposure may cause lung damage or pulmonary sensitization

No data No data No data

Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache

No data

Flush skin/eyes w/ large amounts of water. If inhaled, remove to fresh air. Ingestion: give large amo

of water or milk. Do not induce vomiting.

Evacuate area. Wear SCBA, rubber boots and rubber gloves. Mop up w/ absorptive material and burnchemical incinerato equipped w/ an afterburner and scrubber.

Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalat ion.

Strong sensitizer

NIOSH/MSHA approved respirator

No Chem fume hood

No None

Rubber Splash-proof goggles

Evonik_Agarose Gels for Proteins

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Transcript of Evonik_Agarose Gels for Proteins