Electrophoretic Techniques Lecture 5 PDF

Biomedical Techniques CLSB-222 Dr. Ahmad Alamri, M.H.S, Ph.D. Assistant professor of clinical laboratory sciences [email protected] ELECTROPHORETIC TECHNIQUES  Electrophoresis is defined as the migration of charged molecules in an electric field It is the most commonl...

Biomedical Techniques CLSB-222 Dr. Ahmad Alamri, M.H.S, Ph.D. Assistant professor of clinical laboratory sciences [email protected] ELECTROPHORETIC TECHNIQUES  Electrophoresis is defined as the migration of charged molecules in an electric field It is the most commonly used technique in the clinical laboratory for isolation and quantitation of serum proteins, isoenzymes, hemoglobin, lipoproteins, etc.  Migration of charged molecules is affected by: Net electric charge on the molecules. (pH and Ionic strength of buffer) FACTORS AFFECTING Size and shape of molecules ELECTROPHOR ESIS: Electric field strength Nature of supporting media Temperature of operation  A) Paper Electrophoresis It is the form of electrophoresis that is carried out on filter paper This technique is useful for separation of small charged molecules such as amino acids and small proteins Samples are spotted on filter paper, high voltage is applied, charged molecules migrate towards either the positive or negative pole according to their charge After electrophoresis, separated components can be detected by variety of staining methods, depending on the or chemical identity  B) SDS-Polyacrylamide Gel Electrophoresis Sodium Dodecyl Sulfate-Polyacrylamide gel Electrophoresis (SDS-PAGE), is a technique widely used in biochemistry ,forensics, genetics and molecular biology to separate proteins. This method separates proteins based primarily on their molecular weights.  Principle: Sodium Dodecyl Sulphate [SDS]: is a detergent which denature proteins by binding to the hydrophobic regions, all non-covalent bonds will disrupted and the proteins acquire a negative net charge. So, the proteins samples are having uniformed structure and charge  the separation will depend on their molecular weight only. Small proteins migrate faster through the gel under the influence of the applied electric field, whereas large proteins are successively retarded, due to the sieving effect of the gels. The number of SDS molecules that bind is proportional to the size of the protein Thereby in the electrical field, protein molecules move towards the anode (+) and separated only according to their molecular weight.  During PAGE, the rate of migration of SDS-treated proteins is effectively determined by molecular weight. Polyacrylamide gel: The polyacrylamide gel is formed by co-polymerization of acrylamide and a cross- linking by N,N’-methylene- bis-acrylamide ” bis- acrylamide “. To polymerize the gel a system, consisting of ammonium persulfate (initiator) and tetramethylene ethylene diamine (TEMED) is added[catalyst]. Stain the gel using staining De-stain the gel using De- buffer : staining buffer: It contains: It contains: -Glacial acetic acid -Glacial acetic acid -Methanol -Methanol -Coomassie brilliant blue 250-R  C) Agarose Gel Electrophoresis A technique used to separate DNA fragments based on size using a gel made of agarose. Separate DNA on the basis of their rate of movement through a gel under the influence of an electrical field. Also used to determine the presence and size of PCR products.  DNA is negatively charged. When placed in an electrical field, DNA will migrate toward the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by size. H O2   DNA - + Power Polymerized agarose is porous, allowing for the movement of DNA Scanning Electron  Micrograph of Agarose Gel How fast will the DNA migrate? strength of the electrical field, buffer, density of agarose gel… Size of the DNA! Small DNA move faster than large DNA Gel electrophoresis separates DNA according to size DNA small large - + Power Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.  Sample Preparation Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells. 6X Loading Buffer:   Bromophenol Blue (for color)  Glycerol (for weight)  Carefully place the pipette tip over a well and gently expel the sample. Loading The sample should sink into the well. the Gel Be careful not to puncture the gel with the pipette tip.  Running the Gel Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.  Cathode (-)  wells DNA  Bromophenol Blue (-)  Gel Anode (+) After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.  DNA Ladder Standard - -  12,000 bp  5,000 DNA Note: Bromophenol blue migration  2,000 migrates at approximately  1,650 the same rate as a 300 bp DNA molecule.  1,000  850  650  500  400 bromophenol blue  300  200 +  100 Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.  Staining the Gel Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run. ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times. Staining the Gel Place the gel in the staining tray containing warm diluted stain. Allow the gel to stain for 25-30 minutes. To remove excess stain, allow the gel to destain in water. Replace water several Ethidium Bromide requires an ultraviolet light source to visualize UV Trans illuminator  D) SOUTHERN BLOTTING A Southern blot is a method used in molecular biology for detection of a specific DNA sequence in DNA samples: 2. Size-separation of the 4. Transfer of denatured 1. Digestion of genomic 3. In situ denaturation of fragments (standard DNA fragments into a solid DNA with Restriction the DNA fragments (by agarose gel support (nylon or enzyme  DNA fragments incubation @ ↑temp) electrophoresis) nitrocellulose). 7. Estimation of the size & number of the bands 6. Detection of the bands 5. Hybridization of the generated after digestion complementary to the immobilized DNA to a of the genomic DNA w/ probe (e.g. by labeled probe (DNA, RNA) different RE  placing the autoradiography) target DNA within a context of restriction sites)  E) Northern Blotting Northern Hybridization A northern blot is a method routinely used in molecular biology for detection of a specific RNA sequence in RNA samples.  Basic Steps of Northern Blotting 1. Isolation of intact mRNA 2. Separation of RNA according to size (through a denaturing agarose gel e.g. with Glyoxal/formamide) 3. Transfer of the RNA to a solid support 4. Fixation of the RNA to the solid matrix 5. Hybridization of the immobilized RNA to probes complementary to the sequences of interest 6. Removal of probe molecules that are nonspecifically bound to the solid matrix 7. Detection, capture, & analysis of an image of the specifically bound probe molecules.  F) Western Blotting “Immunoblotting” Applications: To determine the molecular weight of a protein (identification) To measure relative amounts (quantitation) of the protein present in complex mixtures of proteins that are not radiolabeled (unlike immunoprecipitation) Used in medical diagnostics, e.g., in the HIV test or BSE-Test. The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample Advantages: WB is highly sensitive technique Ability to detect picogram levels of protein in a sample  Western Blotting “Immunoblotting” Electrophoretic transfer of proteins from gels to membranes Blotting is the transfer of separated proteins from the gel matrix into a membrane, e.g., nitrocellulose membrane, using electro- or vacuum-based transfer techniques