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Biochemical Practical Methods ADaniyal Ashrafkumar Yuval Amichay Table of Contents 01 Separation02 Analytical methods Methods Immunological Genomic 03 methods 04 methods Separation Methods - Techniques used to separate substances based on certain molecular characteristics - Most important examples in the MIMS course are chromatography and gel electrophoresis Chromatography - Technique used for the separation of mixtures - In MIMS, chromatography methods are often used in the separation of protein mixtures Affinity Chromatography Ion-Exchange Chromatography Gel Filtration Chromatography Affinity Chromatography - Separation of proteins on the basis of affinity to a complementary protein - Stationary phase contains ligand that binds to the protein of interest - Other proteins are washed away when sample is passed through the column - Protein of interest is eluted using a higher affinity competitive ligand Ion-Exchange Chromatography - Separation of proteins on the basis of charge - Stationary phase contains an ion-exchange resin with charged functional groups - Peptides are zwitterionic, so their charge is altered by buffer pH - Proteins with different charges are eluted using a salt gradient: proteins with a higher charge will elute at higher salt concentrations. Gel Filtration Chromatography - Separation of proteins on the basis of size (or more accurately hydrodynamic radius) - Stationary phase contains porous gel beads - “Smaller” proteins become trapped in the stationary phase inside the porous beads - “Larger” proteins are exclude from beads and are eluted first Gel Electrophoresis - Technique used for the separation of macromolecules on the basis of their size and charge by using an electric field - In MIMS, gel electrophoresis is used to separate nucleic acids and proteins Agarose Gels Native PAGE Denaturing PAGE Agarose Gels - Used primarily in the separation of nucleic acids (negatively charged backbone, move to the anode) - The percentage of the gel (e.g. 0.5% gel) refers to the proportion of agarose dissolved in electrophoresis buffer. Higher percentage gels are used for smaller DNA fragments (influence pore size) - Ethidium bromide is the visualizing agent for agarose gels (fluoresces under UV light when intercalated with nucleic acids). - Smaller fragments move with less resistance through the gel. Native PAGE - Used primarily in the separation of Question: what can you conclude proteins, or very small DNA from native gels A and B? fragments - In native PAGE, protein samples are prepared in a non-denaturing and non-reducing buffer. - Separation is based on size AND structure (therefore specific Mr of protein can’t be determined) - However, native PAGE can be used for probe against the protein IRF3 enzyme-linked assays or to study dimerization. - Stained with Coomassie Blue Denaturing PAGE - Same as Native PAGE, but macromoleculares are denatured. - SDS is an anionic detergent used to denature proteins. SDS linearizes the protein and coats it in a uniform negative charge (constant charge to mass ratio). - Mercaptoethanol is also often used to reduce disulphide bonds and further denature proteins. - Urea is commonly used as the denaturing agent for nucleic acids. - Denaturing cause multimeric proteins to dissociate into monomers, but allows you to separate proteins solely on the basis of mass. Analytical Methods - There are a number of techniques used in biochemistry to analyse experimental samples and their contents - In MIMS this year, you will cover a number of these but the most important examples are below Blots - Western, Northern and Southern Protein Assays - Bradford and ELISAs Beer-Lambert Law Western Blots - Used to detect the presence of specific proteins, often from a protein mixture - SDS PAGE is used to initially separate proteins - Proteins are then transferred from the gel to a polymer sheet membrane - This membrane can be exposed to a primary antibody and a secondary antibody conjugated to an enzyme - If the specific protein is present, the coupled enzyme can react with a substrate creating a colour which can be seenWestern Blot Interpretation Samples of serum separated by SDS-PAGE and analyzed by Western Blotting against 10 HIV proteins: gp160, gp120, p66, p55, p51, gp41, p31, p24, p17 and p15. The The numbers in each protein name represent the molecular weight of the protein when resolved by SDS-PAGE electrophoresis in kilodaltons (kDa) Question: With reference to samples from infected individuals, which 4 antigens would you chose as reliable indicators for infection? Answer: p24, p31, p41 and p55Northern and Southern Blots - Southern blots are used to detect a specific DNA sequence from a sample of DNA - DNA first undergoes separation by size using gel electrophoresis - DNA is then transferred to a positively charged membrane to which it covalently attaches - Can use a complementary radioactively tagged DNA probe to see whether a desired piece of DNA is present - Northern blots use the same method but are used to detect RNA instead to determine how much a gene is expressedBradford Assay - Used to determine the total protein concentration in a solution - Coomassie Brilliant Blue G250 binds to certain amino acid side-chains (aromatic, histidine and arginine) - Changes from green-brown in acidic solutions to blue when bound to protein - Absorbance of yellow-orange light (595nm) is proportional to protein concentration - Use calibration curve to work out concentration of protein in sample - However, it is not specificEnzyme Linked Immunosorbent Assay (ELISA) - ELISAs use antibodies to detect the amount of a specific protein present in a sample - Direct ELISA only uses a single primary antibody whereas indirect also uses a secondary antibody - Indirect is highly sensitive and more flexible than direct, however cross-reactivity can occur between the secondary antibody to the adsorbed antigen leading to a non-specific signal occurring - Sandwich ELISAs are flexible, sensitive and are highly specific but, again, cross-reactivity can be a problem.Beer-Lambert Law -Worked Example Immunological Methods - Biochemical methods based on antibody-antigen interactions - Typically antibodies are used to detect specific proteins in a sample (i.e. in ELISA) Immunofluorescence Immunoprecipitation Immunohistochemistry Immunofluorescence - Fluorescently-linked antibodies are used to label proteins within a cell - Proteins may be tagged with an epitope (e.g. Flag), which is useful for visualizing proteins without an antibody. - In indirect immunofluorescence, a Question: Can anyone suggest what primary antibody will bind is being fluorescently tagged in this image of a cell? to the tag, and a secondary fluorescently-labelled antibody binds to the primary antibody. Immunoprecipitation - Specific proteins are precipitated from a sample using an antibody - Antibodies may be immobilized on beads (direct) or beads coated in protein A/G may be added after the antibody is added (indirect) - Precipitated proteins are eluted from the antibodies (using SDS) and analyzed on, for instance, Western Blots via SDS-PAGE - May also lead to co-immunoprecipitation, which can be used to build a protein interactome. Immunohistochemistry (IHC) - Refers to the use of antibodies to identify specific proteins in a biological tissue section - IHC is often used in detecting abnormal cells (i.e. cancer cells) in tissues (specific molecular markers are characteristic of cell death) - IHC can identify markers using immunofluorescence or chromogenic immunohistochemistry (enzyme-linked antibody that catalyzes a color change) Genomic Methods - Methods that are used to study the structure, function and editing of genomes - Enables identification of the genome and comparison between the genomes of different organisms - In MIMS, the main genomic methods you will come across include: Polymerase Chain Reaction (PCR) DNA Sequencing FISH DNA Footprinting Polymerase Chain Reaction (PCR) - Technology that amplifies a copy of DNA to produce millions of copies of a particular DNA sequence - Useful in the lab for purposes such as cloning, sequencing. Also used in forensic analysis and archaeology - Steps: - Denaturing - 2 DNA strands are separated through breaking of hydrogen bonds - Annealing - Primers bind to target DNA sequence - lower temperature - Extension - Taq polymerase joins free nucleotides together and 2 sequences of target DNA formed DNA Sequencing (Sanger) - Sanger sequencing is used to determine the nucleotide sequence of DNA - Developed by Frederick Sanger and his team in 1977 at Cambridge - Steps: - Four separate reaction mixtures each with one of four ddNTPs - DNA chain extended by polymerase until chain is terminated by ddNTP - Gel electrophoresis used to determine relative positions of different bands - The ddNTPs are fluorescently labelled and a chromatograph is produced to identify different nucleotides Fluorescence in situ (FISH) - A type of cytogenetic technique - testing samples such as blood or tissue to identify changes in chromosomes e.g. deletions - Can locate specific RNA targets in cells to understand gene expression patterns within cells DNA Footprinting - Method used to determine protein binding regions on a DNA molecule - Steps: - Two DNA samples - one without protein and the other with suspected protein - DNA treated with nuclease that only digests unprotected DNA - Resulting products separated using SDS PAGE - In sample with protein, the binding regions are protected so these sections are not degradedThank you for your attention!