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Tutorial - Spectrophotometry
1
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5 M to achieve a final volume of 1 L with a concentration of 0
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How many ml of
the stock solution do you need to use? (20mL)
2
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85 at 400 nm, and the molar extinction coefficient (ε) for
the substance at that wavelength is 10,000 L/mol·cm
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(8
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You are studying an enzyme reaction using spectrophotometry
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2 to 0
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The molar extinction coefficient for NADH at
340 nm is 6
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If the path length is 1 cm, calculate the rate of NADH
consumption in µmol/min
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5 μmol/L)
4
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A standard curve
was constructed, and the equation obtained is y=0
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05, where y is absorbance and x
is the protein concentration in mg/mL
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75,
calculate the protein concentration in the sample
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13mg/mL)
5
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The
absorbance of your DNA standards at 260 nm is as follows:
Concentration (µg/mL)
10
20
30
40

Absorbance (260 nm)
0
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41
0
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81

Using the data as above,
a) Plot a standard curve, calculate the slope of the standard curve
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52
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Spectrophotometry relies on the Beer-Lambert law, which states that absorbance is
proportional to the concentration of a substance
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The solute behaves as a perfect absorber at a specific wavelength
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g
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1

In biological samples, deviations may occur due to:
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•
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Turbidity or scattering from particulate matter like cells or proteins
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Absorption by contaminants in the sample, leading to inaccurate measurements
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7
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Prepare a standard curve using known concentrations of a standard protein (e
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, BSA
for the Bradford assay)
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Controls to include:
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•
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A blank sample (buffer without protein) to account for any background absorbance
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Replicates of each sample to ensure accuracy and account for variability
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8
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Explain how could this impact your study
of a compound in a complex biological mixture?
The choice of wavelength is critical for both sensitivity and specificity
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Specificity is achieved by selecting
a wavelength that minimizes interference from other compounds in the mixture
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For example, in a protein-DNA complex, proteins and
nucleic acids absorb at different wavelengths (280 nm for proteins, 260 nm for nucleic
acids)
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To mitigate
this, selecting a wavelength unique to the compound of interest or using dual-wavelength
techniques can help isolate the desired signal
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You are using spectrophotometry to study the interaction between a drug and a target
protein
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Explain how would you determine
the binding affinity of the drug for the protein, and what considerations should you consider
when interpreting the data?
To ensure that absorbance reflects the enzyme reaction rate:
•
•
•

Linear range of the assay: Ensure that the enzyme reaction is within the linear range
of both enzyme and substrate concentrations to avoid saturation effects
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Time intervals: Measure absorbance at appropriate intervals to capture the reaction
kinetics without missing key data points
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Enzyme stability: Ensure enzyme stability over the course of the experiment by
maintaining optimal temperature and pH
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10
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To determine binding affinity, you could perform a binding assay where you vary the
concentration of the drug while keeping the protein concentration constant
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Plot the absorbance versus drug
concentration and fit the data to a binding model (e
...
, Langmuir isotherm) to determine
the dissociation constant (Kd), which reflects the binding affinity
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Stoichiometry: Determine whether the binding is 1:1 or involves multiple binding sites
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Signal saturation: Ensure that absorbance readings do not saturate at high drug
concentrations, which can lead to inaccurate Kd calculations

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