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Bio Practical 2
Next two labs
| Question | Answer |
|---|---|
| Four main classes of Biomolecules and what we focus on | Lipids, Carbs, Proteins, Nucleic Acids We focus on Amino acids and Proteins |
| Test rely on what four characteristics | 1. Hydrophilic or Hydrophobic 2. Color formation with specific reagent 3. Separation based on physical properties (size/ionic charge) 4. Absorption of specific wavelength of light |
| What is Paper Chromatography | Analysis and identification of Amino Acids. Requires a chromatography and matrix. |
| What is the chromatography and matrix used | Chromatography: properties of amino acids used to separate and identify the Amino acids involved spotting samples on Matrix (Chromatography paper) and allow it to dry. Lower edge of paper is exposed to solvent (h20, alcohol etc) in closed container. |
| Absorbed and adsorbed in this experiment | As the solvent is ABsorbed it travels up the matrix, as evidenced by solvent front. Various components of sample are ADsorbed by matrix and move at various rates depending on their degree of aDsorption to matrix and their solubility to solvent |
| What do you do once the solvent front nears upper edge of matrix? | The chromatogram is removed from the solvent and leading edge is marked on the sheet. |
| If the sample is colorless products then what? | Must expose the chromatogram to a chemicle that enables the individual components to be visualized. Ninhydrin often used for amino acid chromatography. Each spot is circled and line drawn thru the middle. Measure origin to mid and mid to solv front |
| What is Rf value | distance traveled by compenent (cm) divided by distance traveled by solvent (cm) |
| Steps in use of | Draw origin 2 cm up with 4 X and labels on top 2. Let spots dry ( pipet 2.5 ul of sample) 10 min 3. Place in jar (20 ml of solvent; keep lid on; origin above solv, 60m 4. T.A. dip in Ninhydrin; dry excessl hang on pin both ends; dry see color |
| Quantitative Analysis of a Protein; Biuret Test | Biuret reaction: color test specific for proteins and small peptides. Contains copper sulfate (blue in solution) copper (I) ions interact with Nitrogen atoms in peptide bonds; cause change in color of solution. |
| Colors formed by Biuret Test | When mixed with biuret reagent (blue) Short peptides cause solution to turn pink Long peptides (Proteins) form complex thats Purple Solutions color and intensity are dependent on type and amount of protein present |
| Our test and our protein tested | We analyze lg protein Albumin expect color Purple. Given "unknow" sample to determine if it has BSA. Obtain tube label "unknown" pipet in 2 ml of distill H20, 2 ml biuret reagent and 1 ml of unknown cap and mix |
| What is our stock | BSA (concent is 2.5 mg/L) BSA standards of diff concentration |
| To choose wavelength | Investigate "absorption spectrum" for biuret reagent/ albumin complex, plot results determine wavelength of max absorbance. Measure all tubes based on wavelength of this, construct St. Curve for BSA to determin concentration in unknown |
| What is an enzyme | Proteins that act as catalysis. Highly specific for the reaction they catalyze, typically grouped according to their function, often clue to which reaction they catalyze. Each cell must have variety of enzymes for multitude of functions. Needed in sm amo |
| Enzyme structure | Only react with specific substrate due to unique conformation and specific "active site" where it binds with it. Enzyme release newly formed products, then avail to react w/ more substrates |
| Whar enzyme are we using | Acid phosphatase (ATP) and its activity under several conditions. Use p-nitrophenyl phosphate for colormetric assay. Amount of product produced correlate to amount of enzyme present |
| types of cell transportation | Osmosis- diffusion of water across a semipermeable cell membrane Diffusion- random mvmt of molecules in response to conct grad high to low Dialysis- diffusions of solutes Active transport- require energy move against their concentration gradient (ATP). |
| What does ATP do in active transport | Drives the transport while a specific membrane protein, carrier molecule, binds to and releases the substance being transported |
| What is Tonicity | the effect of osmosis on living cells. Refers to osmotic pressure (low,equal or high) of a solution relative to the cytosol of living cells. Isotonic, hypertonic, hypotonic |
| How we will study tonicity | Sucrose: non-penetrating solutes, each cell contain equal amount, since each cell has equal amount the external solution will determin whether the cells volume will increase, decrease or stay the same. |
| How do you determine change in each cells volume | By weighing each cell after an incubation period |
| Why must keep tubing moist in experiment | So it wont dry out. Must prep one cell at a time. Dialysis tubing clip bottom pipet sucrose in clip top = one cell Record initial weight |
| Why do we blot the cells dry | blot dry espec. cut ends before weigh, because excess water will affect our results |
| What is dialysis | passive process whereby solutes selectively move across a smipermeable membrane. Only the molecules small enought to pass thru the membrane pores will diffuse, larger ones can not |
| How do we demonstrate dialysis? | We creaste an artificial "cell". Cell membrane is made from dialysis tubing (type of regenerated cellulose that has tiny pores, allow sm to pass lg can not. |
| Our cytosol in experiment | Cytosol is h20, glucose, starch, and albumin. Cells prep by filling dialysis tubing with the cytosol and securely clamping the two ends One cell placed in beaker with distilled h20 Other cell placed in solution of iodine-potassium iodide (I2KI) |
| Based on results we cand determine what | Determine which substances are permeable to the membrane and which are not. |
| Why did content turn blue/black | because of interaction with iodine. Iodine crossed membrane, the starch could not. |
| Why do we test both the original cytosols and water after? | For the presence of glucose and protein |
| What is benedicts test for glucose | colorimetric test for the presence of reducing sugars. Requires samples to be heated in boiling h20. If reducing sugar present, it will react w/ copper sulfate in reagent and "reduced" copper will precipitate out of solution. |
| What will a positive reaction color be | Green, orange, red, depending on the amount of reduced copper. (Reducing sugar is present) |
| Negative reaction color | Blue (NO reducing sugar is present) |
| Biuret test for Protein | Specific for detecting the presenceof proteins in a solution. Contains copper sulfate, potassium hydroxide, and sodium potassium tarrate, Copper ions will readily react with nitrogen atoms, which are present in the peptide bonds of proteins |
| Biuret test blue to purple | The intensity of purple color is related to the amount of protein present. With large proteins, numerous complexes between copper ions and nitrogen atoms can form; the greater the concent. of protein, the deeper color purple. If small peptides then pink |
| Blue vs purple: Biuret test | If albumin is present then stayed blue (negative for proteins) purple (positive for proteins) |
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