Spectrophotometers and Concentration Assays Chapter 7

Objectives Describe how a spectrophotometer works. Differentiate between the different types of spectroscopy. Describe how spectroscopy can be used in measuring protein concentrations. Define pH and describe how it is measured. Describe how a buffer works.

Using Spectrophotometers Spectrophotometry is a method, like others that have been discussed, that can be used to analyze a particular molecule. Allows for the visualization and measurement of a particular molecule in a solution.

Using Spectrophotometers Although a number of different types of spectrophotometers exist all have one thing in common. Utilize light energy to detect molecules in a solution. This light energy is reported to the user as wavelengths in nanometers (nm). Different spec’s utilize wavelengths that fall into different ranges. Visible (VIS) 350-700 nm Ultraviolet (UV) 200-350 nm

Using Spectrophotometers The amount of a particular molecule of interest is measured according to the amount of light that is absorbed. Absorbance data is compared to a standard of a known concentration to determine the concentration of the unknown.

Parts of a Spectrophotometer All spec’s share the following common features: Lamp i.e. tungsten or deuterium Prism or grating Sample holder Display

How a Spec Works User sets the machine to the desired wavelength. Sample is placed in the holder. As light passes through a sample a detector measures the % transmittance. Amount of light passing through a sample that reaches the detector.

How a Spec Works Computer converts the % transmittance to absorbance units (au). Uses Beer’s Law A=2-log10 %T Indirect proportion Must use colored molecules or have a color indicator added. The wavelength that match’s the color of the molecule is not absorbed, all other colors are.

How a Spec Works Known molecules produce spec patterns unique to them. Absorbance spectrum “fingerprint” for a molecule

How a Spec Works How are concentrations obtained using a spec? More molec…more to absorb light Note the peaks of the absorption curves. Lambdamax Wavelength at which a molecule absorbs the most light.

How a Spec Works Enzyme activity can be measured with a spec. Monitor color changes over time. Note changes in absorption peaks.

Using the Spec to Measure Protein Concentrations Proteins, like other molecules, interact with certain wavelengths of light. A proteins absorption spectrum can be determined by measuring the proteins light absorbance at different wavelengths. Determine the lambdamax for the protein.

Using the Spec to Measure Protein Concentrations Most proteins are colorless. Light in the visible range will not work. Light in the UV range will work for a colorless solution. ~280 nm Does NOT distinguish between different protein types in a solution.

Using the Spec to Measure Protein Concentrations Using Bradford Reagent Way to colorize proteins and use white light spectroscopy. Solution changes from brown to blue when proteins present. Degree of “blueness” of Bradford-protein mixture can be used to determine concentration of protein in a solution.

Using the Spec to Measure Protein Concentrations Calculating protein concentration in an unknown sample. Known standards are mixed with Bradford reagent and their absorbance values are determined. Standard curve generated. known absorbance values can be plotted and concentrations determined.

pH Measure of acidity pH= -log [H+] Scale 1-14 Water ionization Table 7.1 pH meters

Maintaining pH - Buffers Aid a solution in resisting change to pH. DNA/proteins must be buffered to maintain their structure and function Salt or organic molecule added to dIH2O. Buffer is prepared at a certain pH (table 7.2) Salt ionizes and interacts with H+ or OH- ions released or added to solution. binds and remove excess

Preparing Buffers Add certain amount of buffering agent to dI H2O Adjust pH down or up using HCL or NaOH to desired value The amount of buffer is determined by the concentration of buffer that is needed

Preparing Buffers Example 1: Example 2: Make 200mL of 0.5M TRIS buffer at pH 7.5 MM TRIS= 121.14g/mol Example 2: Make 500mL of 50mM sodium monophosphate monohydrate buffer at pH=7.5 MM buffer= 137.99g/mol

Buffers Common applications for buffers. Solvent for protein and nucleic acid solutions. Resist pH change in cell culture growth media. Liquid phase in column chromatography (for protein and nucleic acid separation). Liquid for conducting electricity in a gel box.

Homework Review questions section 7.1 page 194 1, 2, 3, 4 Review questions section 7.2 page 198 1, 2, 4 Review questions section 7.3 page 200 1, 2, 3 Think like Biotech page 204 1, 2, 3, 4, 5, 6, 7, 9, 10

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