CYTO_LEC 7_ELECTROPHORESIS 2.pdf

ELECTROPHORESIS Dr. Oliver Smithies LECTURER: Ms. Yellen Eve G. Abarca, RMT British-born American biochemist Nobel Prize in Physiology of Medicine 2007...

ELECTROPHORESIS Dr. Oliver Smithies LECTURER: Ms. Yellen Eve G. Abarca, RMT British-born American biochemist Nobel Prize in Physiology of Medicine 2007 COURSE OUTLINE I. Brief History of Electrophoresis II. Electrophoresis III. Components of Gel Electrophoresis A. Electric Field / Power Supply B. Buffers C. Supporting Medium / Gel Matrices D. Loading Dye or Tracking Dye E. Nucleic Acid Stain F. DNA Ladder G. PCR Products H. Electrophoresis Apparatus Figure 2. Dr. Oliver Smithies IV. Factors Affecting Migration Rate A. Size, Shape, & Net Charge B. Electrical Field Strength Discovered the principle of introducing a specific gene C. Supporting Medium modification in mice using embryonic stem cells. D. Buffer ○ This approach is known as gene targeting. V. Common Problems and How to Avoid Them ○ Through gene targeting, we were able to A. No Bands or Faint Bands discover the disease-causing genes in mice. B. Smearing of the Bands 1950 - used concentrated starches as a matrix in C. Smiling Bands separating serum proteins in blood. D. Poor Separation of Bands 1956 - Dr. Smithies and Dr. Poulik used 2D VI. Other Types of Electrophoresis A. Pulse Field Gel Electrophoresis electrophoresis of serum proteins to describe the B. Capillary Electrophoresis resolution of 20 serum proteins. ○ The high resolution of this method was able to sift proteins at a molecular level, and was LEARNING OBJECTIVES able to differentiate 20 serum proteins in the Discuss the history of electrophoresis. human blood serum. Describe the basic principle of electrophoresis. Identify the components of gel electrophoresis and its functions. Dr. Stellan Hjerten Discuss the electrophoretic process & the common problems. Swedish Biochemist (pupil of Arne Tiselius) encountered in electrophoresis and how to resolve them. I. BRIEF HISTORY OF ELECTROPHORESIS Arne Wilhelm Kaurin Tiselius (Arne Tiselius) Swedish Biochemist, Nobel Prize in Chemistry, 1948 Founding father of Electrophoresis 1930 - published “The Moving Boundary Method of Studying Electrophoresis of Proteins” Returned to Sweden to redesign the electrophoretic U-tube with modifications to resolve complex proteins Figure 3. Dr. Stellan Hjerten better. 1962 - founded his thesis on “A new method of ○ He created it with Dr. Theodor Svedberg, who preparation of agarose for gel electrophoresis” acted like his mentor. ○ Developed the use of agarose gel in electrophoresis, while working with Arne Tiselius. Considered one of the pioneers in Capillary Electrophoresis. University of Uppsala - where Arne Tiselius was able to develop the Electrophoretic U-Tube with Dr. Svedberg. And later on, his own modification of Electrophoretic U-Tube apparatus. Figure 1. Arne Tiselius demonstrating use of an electrophoresis apparatus at the University of Uppsala during a 1939 meeting of the American Chemical Society 1 James W. Jorgenson & Krynn Lukacs Figure 3. James W. Jorgenson & Krynn Lukacs Fig 4. Electrophoresis 1980s - Credited for the introduction of modern Capillary Electrophoresis. III. COMPONENTS OF GEL ELECTROPHORESIS Published “Capillary Zone Electrophoresis” ○ https://doi.org/10.1016/S0301-4770(08)60829-5 In the past, electrophoresis could be performed using solutions. However, it was proven hard to isolate DNA fragments, II. ELECTROPHORESIS especially once the electric field is removed. WHAT IS ELECTROPHORESIS? Recall that Dr. Stellan Hjerten developed a method of using Agarose for Gel Electrophoresis (1962): From the Greek words: For this type of electrophoresis, it uses either agarose ○ Electro - electron or polyacrylamide. In horizontal electrophoresis, the ○ Phoresis - being carried gel impedes the migration of the compound of interest. Migration of charged particles (electrons and protons) The gel is solid but contains microscopic pores or molecules in an electric field: through which electrons and nucleic acids migrate. ○ Cathode Separation of nucleic acid with gel electrophoresis is - negative electrode, attracts positive actually a very reliable method. (cations) Agarose Gel electrophoresis is still widely used to this ○ Anode day. - positive electrode, attracts negative (anions) DISCUSSION - Electrons will migrate here. ○ Can occur in a solution in the electrophoretic U-tube apparatus while further developments also made use of capillary electrophoresis. Separation of charged particles under the influence of an electric field. ○ Biomolecules like nucleic acid (DNA, RNA) are negatively charged because of its sugar-phosphate backbone. Therefore, they migrate to the anode. ○ A lot of significant biomolecules like amino acids and proteins have charges. Performed in tubes, slab gels (vertical or horizontal), or capillaries. They may have different orientations, but they all follow the same principle. The gel contains pores of different sizes, allowing molecules ELECTROPHORESIS OF NUCLEIC ACIDS to migrate to specific points A (large) will reach X Nucleic acids are negatively charged, thus, they B (medium) will reach Y migrate to the positive pole, Anode. C (small) will reach Z Rate of migration will depend on size, shape, and charge as well as overall resistance of the medium. Protein C being smaller, migrates faster than Protein A. It Electric current passes through molecules to move reaches closer to the positive size, indicating its smaller size. them and therefore is separated by the gel. The smaller the protein, the faster it migrates. GEL ELECTROPHORESIS Regardless of power outage or brownout, “ABCs”, or the electrons, will not go back to the cathode. Technique widely used in the field of genetics, biochemistry, and The vibration will always be from the cathode (-) to molecular biology which separates charged molecules on a gel your anode (+). platform under the influence of an electric field. 2 FACTORS THAT WILL AFFECT MIGRATION: B. BUFFER (will be further discussed) Size MAIN FUNCTION: Shape Charge Carries the current. ○ The gel matrix is immersed in the buffer that The shape of your primary protein is straight, so it can go conducts electric current efficiently in relation through the pores. The quaternary structures are arranged to the buffering capacity. tetrahedrally in a three-dimensional structure which means it ○ The buffer must be able to handle the kind of will be harder to migrate fast and through the pores like the electricity needed by the sample. primary protein. A great example of a quaternary protein is the ○ The ions in the buffer help with conductivity Hemoglobin (Hgb). or transmitting electricity. Protects the samples during electrophoresis. Maintains the pH of the medium. A. POWER SUPPLY / ELECTRIC FIELD ○ Since nucleic acids are very sensitive, if the pH of the medium is wrong, it may cause the samples to denature. ○ Controlling the pH helps protect the samples from damage or denaturation. Without the buffer, the gel will burn. COMPONENTS: Contains weak acid and a conjugate base or vice versa. Figure 5. Power Supply A conjugate base is an acid that loses a hydrogen ion. A weak acid with a conjugate base can remain in the Should have constant current, voltage, or power. solution since it doesn’t undergo any type of reaction. A larger separation distance requires a stronger Conjugate base rarely steals a hydrogen proton from electrophoretic field for migration, emphasizing the water. need for a stable power supply. HA + A- → A- + HA If the power supply fails, the compounds will disperse, ○ What will happen if your conjugate base steals and improper electrode function will prevent proper a hydrogen ion? Would it be the same? Does it migration, requiring the experiment to be repeated. change? It won’t change since nothing was removed or added. ELECTRODES (Anode and Cathode) STUDY QUESTIONS QUESTION: What will happen if you increase the concentration of the buffer? What will happen to the conductivity? What is their overall relationship? ANSWER: Increasing the buffer concentration will lead to the buffer conducting more electricity. Therefore, their relationship is directly proportional. Figure 6. Cathode (black) and Anode (red) However, this is not necessarily a good thing. Now that the CATHODE ( – ) ANODE ( + ) buffer can conduct more electricity, it may lead to the Negative electrode Positive electrode denaturation of your samples. Attracts positively charged Attracts negatively charged particles particles CHOOSING A BUFFER: DNA migrates from the sample Increasing the buffer concentration leads to an Attracts DNA wells found here. increased conductivity which can lead to: ○ High voltage which increases heat. Table 1. Cathode vs Anode ○ Increasing heat which makes the molecules If your power cables were not plugged correctly, there move faster, making the gel less stable. will either be no bands or faint bands seen. ○ Less stability which can cause sample denaturation. In this case, the buffer can no longer protect your sample, which is its main purpose, because of too much conductivity. This emphasizes the importance of choosing the correct buffer. The choice of buffer depends on the isoelectric point: 3  pH movement TAE Buffer TBE Buffer isoelectric point no movement Tris base Tris base Glacial acetic acid Boric acid below isoelectric point molecules stay at sample well EDTA EDTA (positively charged) (negative pole) Ideal for large fragments Ideal for small fragments above isoelectric point molecules migrate towards > 1,500 bp < 5,000 bp (negatively charged) positive pole Table 2. pH and movement Not ideal for long runs Ideal for long runs DNA migrates twice as fast DNA migrates slower TYPES OF BUFFERS Has a buffering capacity for longer and higher voltage Table 3. TAE Buffer vs. TBE Buffer SAMPLE WELL At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed. Direction of movement of negatively charged nucleic acids will be from cathode to anode. C. SUPPORTING MEDIUM / GEL MATRIX Figure 7. TAE vs TBE - Molecular Diagram TAE Buffer Tris-Acetate-EDTA contains Tris bases, glacial acetic acid and EDTA ○ Contains a weak acid and conjugate base ○ EDTA acts to prevent the degradation of DNA and RNA and to inactivate nucleases. Nucleases - enzymes that cleave nucleic acids by breaking the phosphodiester bonds. When your PCR product is Figure 8. Gel Matrix under fluorescent light for visualization. contaminated with nucleases, the sample will be denatured and you Provide a matrix where separation takes place. won’t be able to perform Prevents diffusion electrophoresis anymore. ○ In the image above, the bands present are the Ideal for separating larger fragments, cloning, and DNA nucleic acids being separated. If only a extraction from a gel. solution is present without the gel matrix, the Not ideal for long runs. sample would disperse. Ideal for separating large fragments of greater than ○ With the use of a gel matrix, even after the 1,500 base pairs. electrophoretic route is done, the samples DNA migrates twice as fast in TAE compared to TBE. would stay put and not move. This is seen Is often prepared in stock solutions. under UV transilluminators as bands. Forms defined groups. TBE Buffer Tris-Borate-EDTA AGAROSE GEL Contains Tris base, boric acid, and EDTA A polysaccharide polymer from seaweed genera Ideal for resolving small fragments of less than 5,000 Gelidium and Gracilaria. base pairs and ideal for longer runs. Obtained in powdered form which is suspended in a Migration is slower, especially with linear double buffer, heated, and placed in a mold. stranded DNA. ○ This is the reason why you have to prepare Has a buffering capacity for longer or high voltage your running buffer, and dissolve it in the electrophoretic routes. same buffer. ○ In TBE, you can use higher voltage while ○ Dissolving it in a different buffer may result in avoiding sample denaturation. incompatibility and the run will be invalidated. This can also be in the form of tablets that you can prepare in an Erlenmeyer flask or a beaker. 4  Size of spaces in a gel depends on the agarose POLYACRYLAMIDE GEL concentration. ○ The higher the agarose concentration, the Synthetic gel tighter or smaller the pore size. Acrylamide and cross-linker methylene bisacrylamide ○ Agarose concentration and pore size are polymerizes into a matrix. directly proportional. It is inert, electrically neutral, hydrophilic, and transparent for optical wavelengths > 250 nm. FUNCTION: Does not interact with solutes and has low affinity for common protein stains. Molecular Sieve ○ Agarose polymers are non covalent. Instead, What makes this synthetic gel better? they are held together by hydrogen bonds. ○ They form a network of bundles and its pore - It allows for better manipulation size determines the sieving capacity of the - It is possible to tailor the size of the pores gel. - It is better for separating smaller fragments Electrophoresis - Better compared agarose - Electrically neutral AGAROSE GEL CONCENTRATION: - It does not interact with ions in the buffer - It does not interact with solutes How do you determine if your agarose gel concentration is - It has no affinity for common protein stains appropriate? Base it on the size of the DNA you want to resolve: A higher concentration can be achieved than agarose ○ The smaller the DNA you want to resolve, the which is able to separate smaller fragments: more agarose concentration you will need in ○ You have to prepare gradient gels where the order to have smaller and tighter pore size. acrylamide concentration varies from If you are dealing with small DNA and 5% – 20%. your gel concentration is lower, then Small fragments such as ssDNA (single-stranded the DNA will just slip through the large DNA), RNA, and small proteins are best resolved using pores — the bands will not be properly Polyacrylamide Gel Electrophoresis (PAGE) since its separated. pores are smaller. It can also be used with a detergent sodium dodecyl Agarose Concentration (%) Separation range (in bp) sulfate (SDS-Page) which has a strong protein-denaturing effect and binds to the protein 0.3 5,000-60,000 backbone. 0.6 1,000-20,000 Can be used in: 0.8 800-10,000 Nucleic acid sequencing Mutation analyses 1.0 400-8,000 Nuclease protection assays 1.2 300-7,000 ○ Why do we denature protein? Proteins are folded in different 1.5 200-4,000 configurations 2.0 100-3,000 Protein configuration is one of the things which affect the migration of ↑concentration = ↓base pairs charged particles. Relationship: Inversely Proportional This will unfold by denaturing it. Table 4. Agarose Concentration and Separation Range Acrylamide Concentration (%) Separation range (in bp) Generally, 0.3% – 2% agarose concentration is used in large pore size and is ideal for separating nucleic acids 3.5 100 – 1000 and large protein complexes. 5.0 80 – 500 ○ Higher concentration: migration is impeded ○ Lower concentration: not ideal and 8.0 60 – 400 impractical 12.0 40 – 200 The difference between the Agarose gel and the polyacrylamide gel is that it has larger pore size and is 20.0 300 – 7000 ideal for separating nucleic acids and large protein ↑concentration = ↓base pairs complexes. Relationship: Inversely Proportional Human genome project Table 5. Acrylamide Concentration and Separation Range ○ Have 3 billion base pairs all scattered across 46 chromosomes (23 pairs) The indicated figures in the table are referring to gels run in the TBE buffer, since it is better for smaller LECTURER’S NOTE: fragments. No need to memorize the tables as it is only for the purpose of Voltages over 8 V/cm may affect these values. understanding the relationship between the gel concentration Generally, concentration has an inversely proportional and separation range. (Inversely Proportional) with the separation range. 5  ○ But at a certain point, the separation range F. DNA LADDER bottoms out and starts to increase again (at 20% acrylamide, the separation range increases to 300 – 7000 base pairs) D. LOADING DYE / TRACKING DYE Figure 11. DNA Ladder The ladder is used as reference to determine how Figure 9. Tracking Dye many base pairs are present in the DNA being tested. The ladder is standardized. Two primary components: G. PCR PRODUCTS Density Agent (Ficoll, Sucrose, or Glycerol) ○ Denser than the buffer. ○ To prevent diffusion of samples. Tracking Dye ○ Used to monitor the progress of the electrophoresis run. ○ Examples: xylene cyanol FF, bromophenol blue, orange G. ○ There are some commercial mixes such as Promega GoTaq with premixed loading dye. E. NUCLEIC ACID STAIN Figure 12. PCR Product The most important component for electrophoresis is your PCR (Polymerase Chain Reaction) Product. Without this, you won’t have anything to visualize. H. ELECTROPHORESIS APPARATUS Figure 10. Nucleic Acid Stain (SYBR Green) Added to the sample before loading to visualize DNA under a UV or blue light with the use of a UV Transilluminator. A fluorescent stain is added to a gel that binds DNA and fluoresces under UV or blue light. Also called intercalating agents because they stack in between nitrogen bases in double-stranded nucleic Figure 13. Parts of a Gel Electrophoresis Apparatus acid. Parts of the Electrophoresis Apparatus: Examples of Nucleic Acid Stains: Power cables Ethidium Bromide Tank lid ○ Widely used in early DNA and RNA analysis. Casting dams ○ Carcinogenic Gel tray ○ Emit orange light at 300 nm. Electrodes SYBR Green Buffer tank ○ Does not intercalate with the bases but sits in the minor groove of the double helix. ○ Non-mutagenic; safer 6  UV TRANSILLUMINATOR D. BUFFER Determines the direction of electrophoresis: Acidic (positive) → moves to the negative side. Alkali (negative) → moves to the positive side. pH Alkali and Acid electrophoresis: Example: Hemoglobin Figure 14. UV Transilluminators In alkali, the charge of hemoglobin would be negative, so it will migrate to the positive Aids in the visualization of protein and DNA after side. There are some types of hemoglobin electrophoresis. that would be positive or negative depending on the pH of the buffer. Needs to be balanced to get the best resolution. Ionic Strength High electrical conductivity increases the current shared by buffer ions. V. COMMON PROBLEMS DURING ELECTROPHORESIS Figure 15. An example of electrophoresis product (NOTE: absence of a DNA ladder) Figure 16. Faint Bands in Electrophoresis IV. FACTORS AFFECTING MIGRATION RATE FAINT OR NO BANDS CAUSE: SOLUTION: Electrophoretic Mobility - the migration velocity of an ion in a channel under the influence of an electric field. Use gel comb with deep or narrow wells. Sample Quantity Make sure to load an adequate amount of too low The degree of separation of the molecular migration molecules the sample. would depend on these (categorized): Sample degraded Have a nuclease-free work area and (presence of reagents. A. SIZE, SHAPE, AND NET CHARGE nuclease) Continually decontaminate work area. Sample Size Inversely Proportional Voltage may be too high, so decrease the Bigger sample size → slower migration voltage. Shape Smaller sample size → faster migration Sample running Make sure leads are in the proper over the gel orientation. Net Charge Directly Proportional Monitor the run time and migration of the loading dye. B. ELECTRIC FIELD Directly Proportional Voltage High voltage → faster migration Low sample size → slower migration Current Directly Proportional C. SUPPORTING MEDIUM Figure 17. Smeared Bands in Electrophoresis Directly Proportional Pore Size Larger pore size → faster migration SMEARING OF THE BANDS Smaller pore size → slower migration CAUSE: SOLUTION: Inversely Proportional Presence of More agarose (smaller pores) → slower Ensure bubbles are not trapped in the well Agarose Bubbles during migration when loading samples. Concentration Sample Loading Less agarose (larger pores) → faster migration Well might be accidentally punctured during sampling. Well was Do not push the well all the way down the damaged or gel. poorly formed Make sure gel is properly solidifies before wells removing gel comb. Remove the gel comb carefully. 7  Apply voltage as recommended for the VI. OTHER TYPES OF ELECTROPHORESIS Too high or too size range of the nucleic acid. low voltage Do not use more than the recommended PULSE FIELD GEL ELECTROPHORESIS voltage for your sample. Make sure gel preparation and running buffer are compatible. Incompatible Check the buffering capacity. Buffer ○ When using higher voltage, choose a buffer with higher buffering capacity. Figure 20. Pulse Field Gel Electrophoresis A variation of Agarose gel Electrophoresis (AGE) used Figure 18. Anomalous separation or irregular band migration pattern to resolve the large pieces of DNA that cannot be Smiling Bands in Electrophoresis resolved properly by AGE. SMILING BANDS ○ If you wish to resolve larger DNA fragments CAUSE: SOLUTION: higher than the recommended size for AGE, you can use Pulse Field gel electrophoresis Voltage used is Do not use more than the recommended instead. too high voltage. The electric field is alternated between distinct pairs of Choose a buffer that has a high buffering electrodes. capacity. ○ In Pulse Field electrophoresis, the direction of ○ An incompatible buffer won’t be able migration will be different (not in one direction Excessive heat like AGE). to distribute heat evenly. It may be that some parts of the gel heat up faster ○ Electric field periodically changes direction than others. and is sent through pulses. This makes for much clearer bands. Incompatible Ensure that gel is prepared with the same Can separate up to 10 megabases of DNA fragments. buffer buffer used as the running buffer. In microbiology, PFGE is used as a standard method for typing of bacteria. CAPILLARY GEL ELECTROPHORESIS Figure 19. Poor Separation of Bands POOR SEPARATION OF BANDS CAUSE: SOLUTION: Use correct gel percentage for resolving Gel Percentage is the desired size of fragments. incorrect ○ Smaller fragments = higher gel percentage. Figure 21. Capillary Gel Electrophoresis Choose the proper gel type. Gel type used is ○ Polyacrylamide gel is better when A thin glass capillary (fused silica) is used to separate suboptimal working with smaller DNA fragments. the analyte. Do not use more than the needed amount. Originally used for molecules in a solution where the Too much sample Would result in trailing smears or separation was based on size and charge. is used U-shaped bands. A polymer is placed inside the capillary which impedes nucleic acid migration in accordance to size than the charge. 8  REFERENCES https://doi.org/10.1038/npg.els.0005335 Monte S. Willis, Arne Tiselius: Clinical Chemistry, Addgene: Protocol - How to Run an Agarose Gel. (n.d.). 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