module 3

light: conventional and fluorescent - uses visible light to illuminate object - can see most prokaryotic cells and organelles inside eukaryotic cells - objects less than 200nm cannot be seen electron microscopy: transmission and scanning - can visualize small bacteria, viruses, or even large proteins, protein complexes, ribosomes, lipids or small molecules -uses beam of electrons to illuminate object

is the minimum ‘distance’ between two objects that can be distinguished from one another small D = better resolution (see more details) resolution: the ability to see an object (or the amount of detail we observe, high res = more detail observed) , limited by wavelength

D = (0.61(λ))/NA (λ) = wavelength of light, shorter wavelengths of light give higher resolution (smaller λ = smaller D) improving resolution NA = numerical aperture - a measure of the ability to gather light and resolve specimen detail - how light is bent/refracted as it passes from the glass of the microscope objective through air to the specimen - the closer NA is to 1, the better

decreasing D lower λ -> lower D = better resolution

visible light λ= 400nm to 700nm D = (0.61(450))/0.94 = 292nm 0.94 = microscope objective 292nm or almost 300nm means that if 2 objects are closer together than 292nm, they cannot be distinguished under the microscope. Limit on size of objects we see w/microscope

UV light: λ = 200nm to 300nm - outside of visible light spectrum - using this light source reduces D, giving better resolution electron microscopy: Uses a beam of electrons, shorter λ - Limit of resolution is 0.1 nm to 10 nm - can distinguish objects that are only 1/10th of a nanometer apart

• samples may be lived or fixed • type of light microscopy • can see shape of sickled/unsickled RBC , mouse cell intestine (surface columnar epithelial cells, goblet cells, nuclei) • samples may be stained or unstained • samples may be embedded in parifin to slide thin sections for observation under the microscope

Nomarski or DIC (differential inference contrast microscopy): clear, sharper images of edges and surfaces of cellular structures - clearer periphery to help study intracellular events and proximity between cells Phase contrast: favours clear cell visualization of internal cellular structures - Halo created around external surface of cell, that obscures some visualization Both - allow live specimens to be visualized, allowing us to look at dynamics of a living system - both rely on enhancing inherent difference in density of different regions of the specimen

• uses light fluorescence to reveal locations of specific molecules in the cell • molecules of interest are stained directly with fluorescent dyes or tagged with fluorescent antibodies • Use a primary antibody to specifically locate a molecule or antigen of interest (to detect antigen B over antigen A for ex) • primary antibody recognizes and binds to protein/antibody of interest. From there, a secondary antibody is used to recognize the primary antibody. This secondary antibody is covalently attached to a fluorescent molecule (flourophore) • Flourophore is excited by UV light so that it emits another wavelength of fluorescent light. This way, we can specifically detect our protein of interest and ignore all the other proteins in the cell.

green: CD44 cell membrane marker blue: DNA stain (DAPI) • allows us to see mitosis steps more clearly (requires cells to be fixed and further processes before further imaging)

GFP - green fluorescent protein • GFP is a naturally derived fluorescent protein originally discovered in jelly fish • using recombinant DNA tech, researchers can create a gene fusion between the gene coating for protein of interest and the gene coating for GFP • this fused recombinant DNA can be reintroduced into cells where the gene can be expressed to make the now fluorescent fusion protein - GFP Advantages - allows us to see the dynamics of spindle assembly and disassembly in the mitotic cell - we can also use it to track synthesis, localization, movement and degradation of any unique protein in the cell

• excites a thin layer of the sample to eliminate background fluorescence for clearer image • higher resolution images • create optical sections while keeping cells and tissue intact by exciting only the flourophores in a thin section of the sample (using a specialized laser) ex: DNA in nucleus stained with DAPI (blue), an actin specific binding protein (phalloidin) is tagged w/the flourophore flourscein to label actin (green). and antibody to visualize the golgi apparatus (shown in red)

• in this technique a fluorescence image (taken with a conventional fluorescence microscope) is processed using computer algorithms to subtract out the fluorescence that is out of focus above and below the focal plane of the image • able to create digital sections of fluorescent images

Beam of electrons directed at sectioned specimen * Limit of resolution of 0.1 nm beam of electrons is directed through a thinly sliced specimen to form an image. stained or dense areas of the sample appear dark, while unstained or sparser areas appear light can see microvilli clearly, more structure visible at surface and inside of cell

beam of electrons directed at the surface (that has been coated with a layer of metal - to improve electrical conductivity, which prevents charging and image distortion) of solid specimens * Reveals information about surface morphology and texture lets us see more 3D structure

• structural analysis • functional analysis (interacting substrates and what substrate binding does) • determination of amino acid sequence (or find gene that codes for protein) • development of antibodies to a protein (these antibodies can be used in immunofluorescence microscopy to detect location in cell)

• Develop a good assay (experiment) • Select protein source • Break open cells - protein extract (from cell) • Solubilize protein • Stabilize protein • Fractionate (separate your proteins from all other proteins) • Determine purity

Method of detecting for the presence of a specific protein and estimating the concentration of the protein assay should be specific to the protein you are studying and based upon unique characteristics (so you don’t mistake for a diff. protein) Examples: * Enzymatic activity (looking for release of a product or use of substrate) * Immunological assay (antibody) - (antibody used to monitor presence and concentration of protein) * Biological activity/function (if protein binds to biological substrate - RNA, actin..)

• easily obtainable in large amounts (tissue, cultured cells, over expression in another system) • contain a high concentration of the protein of interest (ex: RBC to study hemoglobin) • low in proteins that may co-purify (have similar characteristics to protein of interest) • low in proteases that may destroy the protein of interest (can also add protease inhibitors to make sure proteins are not destroyed during purification) need to break open/lice cells to release all proteins - best to express ur protein in an alternate cell type in order to ensure you’re extracting the greatest amount of your protein of interest as possible

predict whether you expect a protein to be soluble in your extract based upon the properties that you believe it to have soluble: cytosolic or secreted proteins (solubility affected by pH of solution, salt conc, presence of detergents help stabilize molecular interactions in protein which can increase the solubility of insoluble proteins) - by changing extract conditions, you can inc. solubility of any protein insoluble: transmembrane or membrane associated proteins (difficult to isolate cause they are amphipathic w/hydrophobic and hydrophilic properties)

protein must first be stable in order to purify it stabilization maintains native protein structure (optimally folded and functional form) to prevent it from degradation Important Parameters (need to maintain non-covalent interactions that are stabilizing the folded conformation - pH, salt conc, presence of co factors will help maintain native conformation) 1. Temperature (too warm will denature) 2. Protease inhibitors (should be added to cell extract to prevent degradation) 3. Ligands 4. Salts 5. Metal ions 6. Concentration (soluble protein may aggregate if present at a higher conc) 7. pH

process of separating proteins into diff groups or fractions - many diff. techniques used , each takes advantage of a chemical or physical property of protein of interest that distinguishes it from others What criteria can be used to separate the protein of interest from other proteins? How do proteins differ from one another? Proteins vary in (take these into consideration for fractionation) size, polarity, charge, solubility, shape. a single technique will generally not be sufficient for protein isolation, you will need a series of fractionation experiments to isolate protein of interest

• ion exchange chromatography (column containing charged beads. Protein extract loaded at top of column (ex: red - pos, blue - neg). Red will flow through quickly cuz positive charge protein is repelled by the beads. Blue (neg) will be attracted to red (pos) and move more slowly through the column. To elute/remove blue protein from beads: - add salt (NaCl) solution: interactions between neg. charged chloride and the beads will disrupt the interaction between blue protein and beads, allowing blue protein to flow through column - warm wash solution: disrupt association between the beads and proteins - change pH of wash solution • gel electrophoresis

• absorption chromatography • hydrophobic interaction chromatography separation by chromatography: in this technique: an aqueous extract is poured into a column containing a matrix or a slurry of tiny beads that help sort proteins based on diff. properties • diff proteins (pink vs blue for ex), have been separated into diff. fractions upon the degree of interaction w/the beads in the column More Interaction (between proteins and beads) = protein moves very slowly through the column Less interaction = protein moves quickly past the beads to the bottom of the column