Electrophoretic Properties of Native Proteins

Transcription

1 The Biotechnology Education Company 111 EDVO-Kit # Electrophoretic Properties of Native Proteins Storage: See page 3 for specific storage instructions. EXPERIMENT OBJECTIVES: The objective of this experiment is to develop a general understanding of the structure and electrophoretic migration of native proteins. 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.

2 2 Table of Contents Page Experiment Components 3 Experiment Requirements 3 Background Information 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

3 3 Experiment Components Quick Reference: There is enough sample for 6 gels if you are using an automatic micropipet for sample delivery. Use of transfer pipets will yield fewer gels. A B C D E Bovine Serum Albumin (BSA) Ovalbumin Cytochrome C Lysozyme 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 Store Components A-E at 4 C. This experiment contains ready-to-load protein samples and reagents sufficient for 6 gels (see Quick Reference). 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. UltraSpec-Agarose and Protein Plus are trademarks of EDVOTEK, Inc. Protein InstaStain, EDVOTEK, and The Biotechnology Education Company are registered trademarks of EDVOTEK, Inc. Requirements 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 EDVOTEK - The Biotechnology Education Company EDVOTEK 24-hour FAX: (301) edvotek@aol.com

4 4 Properties of Native Proteins Background Information N-Terminal Glutamic Acid Lysine H + H-N-H Proteins are a highly diversified class of biomolecules. Differences in their chemical 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 small molecules, 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 molecule can be electrically neutral, i.e. negative and positive charges are equal. In this case, the protein is isoelectric. In the presence of an electrical field, a protein with a net negative or positive charge will migrate towards the electrode of opposite charge. The amino acid residues responsible for a protein s negative charge at physiological ph are glutamic acid and aspartic acid. A protein s positive charge at physiological ph is due to lysine, arginine and, to a lesser extent, histidine. O CH 2 - CH 2 - C OH H + CH 2 - CH 2 - CH 2 - CH 2 - N - H H H + H-N-H O CH 2 - CH 2 - C OH H + CH 2 - CH 2 - CH 2 - CH 2 - N - H H H + H-N-H O CH 2 - CH 2 - C OH H CH 2 - CH 2 - CH 2 - CH 2 - N H C C-Terminal O HO Acidic ph ( < 4 ) C C O O O - O - Neutral ph ( 7 ) Figure 1: Effect of ph on Peptide Containing Glutamic and Lysine Residues Alkaline ph ( > 9 ) 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 and become 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 protein s migration in an electric field can be altered by using an acidic, neutral or alkaline buffer system during electrophoresis. The isoelectric point of a protein is defined as the ph at which the protein has no net charge. Consequently, a protein will not migrate in an electric field at its (pi) isoelectric point. An isoelectric protein still possesses areas of negative and positive charge, but overall

5 Background Information 5 Properties of Native Proteins they cancel each other. Proteins that contain more aspartic and glutamic 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 amino acid 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 attached or very tightly bound to the polypeptide chain(s) of a protein are called prosthetic groups. Prosthetic groups enable proteins to perform biological functions and have a strong influence on the protein s chemical properties. The oxygen atoms transported by hemoglobin are actually bound at the heme irons. Proteins exhibit many different three-dimensional shapes and complex folding patterns which are determined by their amino acid sequence and post translational processing such as adding carbohydrate residues or prosthetic 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 of the number and type of amino acids in the polypeptide chain. Proteins can consist of a single polypeptide or several polypeptides specifically associated 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 heat can cause a native protein to lose its specific three-dimensional folding pattern and, consequently, its biological activity. Proteins that have gone through this process are called denatured. Denatured proteins can have radically different behavior from their native forms during electrophoresis. ELECTROPHORESIS OF PROTEINS The properties of proteins affect the way they migrate during gel electrophoresis. Gels used in electrophoresis (e.g. agarose, polyacrylamide) consist of microscopic pores of a defined size range that act as a molecular sieve. Only molecules with net charge will migrate through the gel when it is in an electric field. Small molecules pass through the pores more easily than large ones. Molecules having more charge than others of the same shape and size will migrate faster. Molecules of the same mass and charge can have different shapes. In this case, those with a more compact shape, like a sphere, will migrate through the gel more rapidly 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.

6 6 Properties of Native Proteins Background Information Electrophoresis of native proteins is useful in clinical and immunological analysis 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 most abundant 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 regulation. The serum proteins with the slowest migration rates are the gamma globulins (antibodies). In between these two zones several other types of proteins can be observed. These include transferrin (iron transport), ceruloplasmin (copper transport), macroglobulin (protease inhibitor) and haptoglobin (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 in albumin, while multiple myeloma ( a cancer of the immune system) and chronic rheumatoid arthritis causes abnormal increases in the gamma globulins. The purified proteins used in this experiment all consist of single polypeptide 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, is similar in structure and function to the serum albumin previously discussed. Ovalbumin is an abundant protein found in eggs. Ovalbumin, with a molecular 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 also glycoproteins (except serum albumin). Cytochrome C, a ubiquitous protein with a molecular weight of 12,000 and pi of 10.7, is involved in electron transport reactions that are mediated by the heme prosthetic group. Cytochrome C and several other proteins form the electrontransport 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 protein with 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 and the 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 two bands. This separation occurs because some of the serum proteins bind a fraction of the dye. The bound dye has a slower migration rate than the free dye. After electrophoresis, the proteins will be visualized by staining with Protein InstaStain After the gel is destained, proteins will appear as dark blue zones against a light blue background.

7 Experiment Procedures 7 Experiment Overview EXPERIMENT OBJECTIVE: The objective of this experiment module is to develop a general understanding of the structure and electrophoretic migration of native proteins. LABORATORY SAFETY Wear gloves and safety goggles 1. Gloves and goggles should be worn routinely as good laboratory practice. 2. Exercise extreme caution when working with equipment which is used in conjunction with the heating and/or melting of reagents. 3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS OR BULBS.

8 8 Preparations for Agarose Gel Electrophoresis PREPARING THE GEL BED Experiment Procedures Wear gloves and safety goggles 1. Make sure the gel bed is clean and dry. 2. Close off the open ends of the bed by using rubber dams or tape. A. Using Rubber dams: Place a rubber dam on each end of the bed. Make sure the rubber dam sits firmly in contact 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 and bottom. 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 and evenly 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 disperse clumps. 5. With a marking pen, indicate the level of the solution volume on the outside of the flask. Table A Individual 0.8% UltraSpec-Agarose Gel Size of EDVOTEK Casting Tray (cm) Amt of Agarose (g) Concentrated Distilled + Buffer (50x) + Water = Total Volume 7 x

9 Experiment Procedures 9 Preparations for Agarose Gel Electrophoresis 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 60 C with careful swirling to promote even dissipation of heat. If detectable evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step C After the gel is cooled to 60 C: If using rubber dams, go to step 9. If using tape, continue with step 8. Caution! DO NOT POUR BOILING HOT AGAROSE INTO THE GEL BED. Hot agarose solution may irreversibly warp the bed. 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 to solidify. 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 after approximately 20 minutes.

10 10 Preparations for Agarose Gel Electrophoresis Experiment Procedures PREPARING THE SOLIDIFIED GEL FOR ELECTROPHORESIS 11. Carefully remove the rubber dams or tape. 12. Remove the comb by slowly pulling it straight up. 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 wells are 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 experiment instructions. See Table C for time and voltage guidelines. Useful Hint! Table B Electrophoresis Buffer Step 11: Be careful not to damage or tear the gel when removing rubber dams. A thin plastic knife or spatula can be inserted between the gel and the dams to break possible surface tension. EDVOTEK Model # M6+ M12 M36 (blue) M36 (clear) Concentrated Buffer (50x) Distilled Water = Total Volume

11 Experiment Procedures 11 Practice Gel Loading Quick Reference: If you are using an automatic micropipet, deliver 20 microliters to the sample well. If using transfer pipets, load the sample well until it is full. Remember! Use microbiology-grade agar and water to make practice gels - save the agarose for the experiment. EDVOTEK experiments which involve electrophoresis contain practice gel loading solution. If your students are unfamiliar with loading samples in agarose gels, it is suggested that they practice sample delivery techniques before performing the electrophoresis part of an experiment. Using the EDVOTEK system, sample delivery can be performed by using either an automatic micropipet, or disposable microtipped transfer pipets. Casting of a separate practice gel is highly recommended. One suggested activity for practice gel loading is outlined below: 1. Cast a gel with the maximum number of wells and place it under the buffer in an electrophoresis apparatus chamber. (Use microbiology-grade agar and water to make practice gels - save the agarose for the experiment.) 2. Let students practice delivering the practice gel loading solution 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 practice gel with a fresh gel and continue with the experiment. The practice gel loading solution will become diluted in the buffer and will not interfere with the experiment.

12 12 Conducting Agarose Gel Electrophoresis Experiment Procedures Reminder: During electrophoresis, the DNA samples migrate through the agarose gel towards the positive electrode. Before loading the samples, make sure the gel is properly oriented in the apparatus chamber. - + Black Red Sample wells 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 electrophoresis apparatus leads reach the power source before loading samples. The apparatus should not be moved after the samples are loaded because movement of the unit will cause samples to spill out of the wells. LOADING PROTEIN SAMPLES 1. Consecutively load 40µl of each sample in tubes A - E into wells in the middle of the gel. RUNNING THE GEL 2. After the samples are loaded, carefully snap the cover down onto the electrode terminals. M12 Electrophoresis Table C: Volts Recommended Time Minimum Time and Voltage Optimal min 45 min min 1.5 hrs min 2.0 hrs Useful Hint! After electrophoresis, remove a small slice of the gel from the upper right hand corner to easily identify right and left orientation of the gel after staining. Make sure that the negative and positive indicators on the cover and apparatus chamber are properly oriented. 3. Insert the plug of the black wire into the black input of the power source (negative input). Insert the plug of the red wire into the red input of the power source (positive input). 4. Set the power source at the required voltage and run the electrophoresis for the length of time as determined by 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 leads and remove the cover.

13 Experiment Procedures 13 Staining the Gel ONE-STEP STAINING & DESTAINING WITH PROTEIN INSTASTAIN Protein agarose gels can be stained with Protein InstaStain cards in one easy 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 against a 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 bands against the background improves. Fixative and Destaining Solution for each gel (100ml) 50ml 10ml 40ml Methanol Glacial Acetic Acid Distilled Water

14 14 Study Questions Experiment Procedures 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? 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, Lysozyme 14, Ovalbumin 43, BSA 68, All these proteins are spherical in shape, therefore, increasing molecular weight corresponds to increasing size. The results of the electrophoresis at ph 7.8 should show that ovalbumin migrates a greater distance towards the positive electrode than BSA. The results should also show that Lysozyme migrates a greater distance towards the negative electrode than Cytochrome C. Explain the results using the information provided above.

15 15 Notes to the Instructor M o n Online Ordering now available Visit our web site for information about EDVOTEK 's complete line of experiments for biotechnology and biology education. EDVO-TECH - Fri 9 SERVICE EDVOTEK ( ) am - 6 E T pm If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledgeable technical staff at EDVOTEK ( ). 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 the gel(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 2 hours. Technical Service Department Mon - Fri 9:00 am to 6:00 pm ET FAX: (301) web: edvotek@aol.com Please have the following information: The experiment number and title Kit Lot number on box or tube The literature version number (in lower right corner) Approximate purchase date A variety of factors, such as class size, length of laboratory sessions, and availability of equipment, will influence the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. SPECIFIC REQUIREMENTS FOR THIS EXPERIMENT Gel Concentration This experiment requires a 0.8% UltraSpec- Agarose gel. Number of Wells Required This experiment requires a gel with 5 sample wells. Instructor's Guide Placement of Comb During gel casting, the comb should be placed in the notches in the middle of the gel bed.

16 16 PreLab Preparations PREPARING THE ELECTROPHORESIS (CHAMBER) BUFFER Instructor's Guide Useful Hint! The same 50x concentrated buffer is used for preparing the agarose gel buffer and the chamber buffer. Prepare the appropriate volume of diluted electrophoresis chamber buffer by mixing the concentrated 50x electrophoresis buffer and distilled or deionized water according to Table B. ELECTROPHORESIS TIME AND VOLTAGE Your schedule will dictate the length of time samples will be separated by electrophoresis. General guidelines are presented in Table C. PREPARING STAINING AND DESTAINING SOLUTIONS Wear gloves and safety goggles 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 ml Glacial Acetic Acid. No destaining is required.

17 17 Quick Reference Tables Table A Size of EDVOTEK Casting Tray (cm) Amt of Agarose (g) Concentrated Distilled + Buffer (50x) + Water = Total Volume 7 x Table B Individual 0.8% UltraSpec-Agarose Gel Electrophoresis Buffer Instructor's Guide EDVOTEK Model # Concentrated Buffer (50x) + Distilled Water = Total Volume M M M36 (blue) M36 (clear) Time and Voltage Electrophoresis of DNA Table C Volts Recommended Time Minimum Maximum min 40 min 60 min 45 min 1.5 hrs 2.0 hrs

18 18 Avoiding Common Pitfalls Instructor's Guide To ensure that protein bands are well resolved, make sure the gel formulation is correct (see Table A on page 17) and that electrophoresis 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 according to instructions. Before performing the actual experiment, practice sample delivery techniques to avoid diluting the sample with buffer during gel loading. 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 direction 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.

19 19 Idealized Schematic of Results ( - ) The figure to the left is an idealized schematic showing relative 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 Lysozyme 5 E Horse Serum Proteins Instructor's Guide ( - ) ( + ) ( + )

20 Please refer to the kit insert for the Answers to Study Questions

21 Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Stable Incompatibility available X Conditions to Avoid IDENTITY (As Used on Label and List) Agarose Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Other Limits Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional) This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. CAS # Section III - Physical/Chemical Characteristics Boiling Point For 1% solution 194 F Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Evaporation Rate Vapor Density (AIR = 1) (Butyl Acetate = 1) Solubility in Water Insoluble - cold Appearance and Odor White powder, no odor Section IV - Physical/Chemical Characteristics N.D. = Flash Point (Method Used) Flammable Limits LEL N.D. UEL N.D. Extinguishing Media Water spray, dry chemical, carbon dioxide, halon or standard foam Special Fire Fighting Procedures Possible fire hazard when exposed to heat or flame Hazardous Decomposition or Byproducts Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Inhalation: available Ingestion: Large amounts may cause diarrhea Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure available Medical Conditions Generally Aggravated by Exposure available Emergency First Aid Procedures Treat symptomatically and supportively 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 Respiratory Protection (Specify Type) Ventilation Local Exhaust Special Protective Gloves Yes Sweep up and place in suitable container for disposal Normal solid waste disposal Chemical cartridge respirator with full facepiece. Mechanical (General)Gen. dilution ventilationother Eye Protection Splash proof goggles Unusual Fire and Explosion Hazards Other Protective Clothing or Equipment Work/Hygienic Practices Impervious clothing to prevent skin contact Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable X Incompatibility Strong oxidizing agents IDENTITY (As Used on Label and List) 50x Electrophoresis Buffer Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Special Fire Fighting Procedures Unusual Fire and Explosion Hazards identified Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Other Limits Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. % (Optional) Section III - Physical/Chemical Characteristics Boiling Point Specific Gravity (H 0 = 1) 2 Vapor Pressure (mm Hg.) Melting Point Vapor Density (AIR = 1) Evaporation Rate (Butyl Acetate = 1) Solubility in Water Appreciable, (greater than 10%) Appearance and Odor Clear, liquid, slight vinegar odor Section IV - Physical/Chemical Characteristics N.D. = Flash Point (Method Used) Flammable Limits LEL UEL N.D. N.D. Extinguishing Media Use extinguishing media appropriate for surrounding fire. Wear protective equipment and SCBA with full facepiece operated in positive pressure mode. Hazardous Decomposition or Byproducts Carbon monoxide, Carbon dioxide Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Carcinogenicity: identified NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Irritation to upper respiratory tract, skin, eyes Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures Ingestion: If conscious, give large amounts of water Eyes: Flush with water Inhalation: Move to fresh air Skin: Wash with soap and water Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Wear suitable protective clothing. Mop up spill and rinse with water, or collect in absorptive material and dispose of the absorptive material. Waste Disposal Method Dispose in accordance with all applicable federal, state, and local enviromental regulations. Precautions to be Taken in Handling and Storing Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust Yes Special Mechanical (General) Yes Other Protective Gloves Yes Eye Protection Safety goggles Other Protective Clothing or Equipment Work/Hygienic Practices Avoid eye and skin contact.

22 Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section V - Reactivity Data Stability Unstable Stable Incompatibility X Conditions to Avoid IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must Practice Gel Loading Solution be marked to indicate that. Section I Manufacturer's Name Emergency Telephone Number (301) EDVOTEK, Inc. Telephone Number for information Address (Number, Street, City, State, Zip Code) (301) Rothgeb Drive Rockville, MD Date Prepared Signature of Preparer (optional) Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Section III - Physical/Chemical Characteristics Boiling Point Vapor Pressure (mm Hg.) Vapor Density (AIR = 1) Solubility in Water Specific Gravity (H 0 = 1) 2 Melting Point Evaporation Rate (Butyl Acetate = 1) Other Limits Recommended This product contains no hazardous materials as defined by the OSHA Hazard Communication Standard. Soluble % (Optional) Hazardous Decomposition or Byproducts Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Yes Skin? Yes Ingestion? Yes Health Hazards (Acute and Chronic) Acute eye contact: May cause irritation. available for other routes. Carcinogenicity: NTP? IARC Monographs? available OSHA Regulation? Signs and Symptoms of Exposure May cause skin or eye irritation Medical Conditions Generally Aggravated by Exposure reported Emergency First Aid Procedures Treat symptomatically and supportively. Rinse contacted area with copious amounts of water. Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled Wear eye and skin protection and mop spill area. Rinse with water. Waste Disposal Method Observe all federal, state, and local regulations. Precautions to be Taken in Handling and Storing Avoid eye and skin contact. Sulfur oxides, and bromides Appearance and Odor Blue liquid, no odor Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL UEL Extinguishing Media Dry chemical, carbon dioxide, water spray or foam Special Fire Fighting Procedures Use agents suitable for type of surrounding fire. Keep upwind, avoid breathing hazardous sulfur oxides and bromides. Wear SCBA. Unusual Fire and Explosion Hazards Unknown Other Precautions Section VIII - Control Measures Respiratory Protection (Specify Type) Ventilation Local Exhaust Yes Special Mechanical (General) Yes Other Protective Gloves Yes Eye Protection Other Protective Clothing or Equipment required Work/Hygienic Practices Avoid eye and skin contact Splash proof goggles IDENTITY (As Used on Label and List) Protein Plus Stain Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Methanol (Methyl Alcohol) 200ppm 200ppm 90%-100% CH3OH Other Limits Recommended % (Optional) Section III - Physical/Chemical Characteristics Boiling Point 65 C Specific Gravity (H 0 = 1) 2.79 Vapor Pressure (mm Hg.) 96mmHg Melting Point N/A Vapor Density (AIR = 1) 1.11 Evaporation Rate (Butyl Acetate = 1) 4.6 Solubility in Water Complete (100%) Appearance and Odor Blue liquid/alcoholic, pungent odor Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL UEL (closed cup) 12 C 6.0% 36% Extinguishing Media Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective) Special Fire Fighting Procedures Wear SCBA with full facepiece operated in positive pressure mode. Move containers from firearea Unusual Fire and Explosion Hazards Vapors may flow along surfaces to distant ignition sources. Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire. Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable X Incompatibility Strong oxidizing agents Hazardous Decomposition or Byproducts Carbon monoxide, Carbon dioxide, Sulfur oxides Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Skin? Ingestion? Yes Yes Yes Health Hazards (Acute and Chronic) Irritating to eyes, skin, mucous membranes and upper respiratory tract. Chronic exposure may cause lung damage or pulmonary sensitization Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures 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. Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled 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. Waste Disposal Method Observe all federal, state, and local laws. Precautions to be Taken in Handling and Storing Wear protective gear. Avoid contact/inhalation. Other Precautions Strong sensitizer Section VIII - Control Measures Respiratory Protection (Specify Type) NIOSH/MSHA approved respirator Ventilation Local Exhaust No Special Chem fume hood Mechanical (General) No Other Protective Gloves Rubber Eye Protection Splash-proof goggles Other Protective Clothing or Equipment Rubber boots Work/Hygienic Practices Avoid prolonged or repeated exposure

23 IDENTITY (As Used on Label and List) Protein InstaStain Section I Manufacturer's Name EDVOTEK, Inc. Address (Number, Street, City, State, Zip Code) Rothgeb Drive Rockville, MD Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication Standard. 29 CFR Standard must be consulted for specific requirements. Section II - Hazardous Ingredients/Identify Information Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that. Emergency Telephone Number (301) Telephone Number for information (301) Date Prepared Signature of Preparer (optional) Methanol (Methyl Alcohol) 200ppm 200ppm 90%-100% CH3OH Other Limits Recommended % (Optional) Section III - Physical/Chemical Characteristics Boiling Point 65 C Specific Gravity (H 0 = 1) 2.79 Vapor Pressure (mm Hg.) 96mmHg Melting Point N/A Vapor Density (AIR = 1) 1.11 Evaporation Rate (Butyl Acetate = 1) 4.6 Solubility in Water Complete (100%) Appearance and Odor chemical bound to paper, no odor Section IV - Physical/Chemical Characteristics Flash Point (Method Used) Flammable Limits LEL UEL (closed cup) 12 C 6.0% 36% Extinguishing Media Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective) Special Fire Fighting Procedures Wear SCBA with full facepiece operated in positive pressure mode. Move containers from firearea Unusual Fire and Explosion Hazards Vapors may flow along surfaces to distant ignition sources. Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire. Section V - Reactivity Data Stability Unstable Conditions to Avoid Stable X Incompatibility Strong oxidizing agents Hazardous Decomposition or Byproducts Carbon monoxide, Carbon dioxide, Sulfur oxides Hazardous May Occur Conditions to Avoid Polymerization Will Not Occur X Section VI - Health Hazard Data Route(s) of Entry: Inhalation? Skin? Ingestion? Yes Yes Yes Health Hazards (Acute and Chronic) Irritating to eyes, skin, mucous membranes and upper respiratory tract. Chronic exposure may cause lung damage or pulmonary sensitization Carcinogenicity: NTP? IARC Monographs? OSHA Regulation? Signs and Symptoms of Exposure Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache Medical Conditions Generally Aggravated by Exposure Emergency First Aid Procedures 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. Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled 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. Waste Disposal Method Observe all federal, state, and local laws. Precautions to be Taken in Handling and Storing Wear protective gear. Avoid contact/inhalation. Other Precautions Strong sensitizer Section VIII - Control Measures Respiratory Protection (Specify Type) NIOSH/MSHA approved respirator Ventilation Local Exhaust No Special Chem fume hood Mechanical (General) No Other Protective Gloves Rubber Eye Protection Splash-proof goggles Other Protective Clothing or Equipment Rubber boots Work/Hygienic Practices Avoid prolonged or repeated exposure