Podcast
Questions and Answers
Which statement about colorimetric assays is correct?
Which statement about colorimetric assays is correct?
What change occurs in Coomassie blue dye during the Bradford Assay?
What change occurs in Coomassie blue dye during the Bradford Assay?
What is indicated by a high R^2 value on a standard curve analysis?
What is indicated by a high R^2 value on a standard curve analysis?
Which component is NOT part of the Beer-Lambert Law equation?
Which component is NOT part of the Beer-Lambert Law equation?
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In a dilution problem, if you have 100 µL of a 1 mg/mL protein sample and dilute it to a total of 500 µL, what will the final concentration be?
In a dilution problem, if you have 100 µL of a 1 mg/mL protein sample and dilute it to a total of 500 µL, what will the final concentration be?
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Which of the following is a common error that can affect the accuracy of measurements in colorimetric assays?
Which of the following is a common error that can affect the accuracy of measurements in colorimetric assays?
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How can you ensure that your standard curve is valid?
How can you ensure that your standard curve is valid?
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What is the consequence of not achieving equilibrium in early-stage reactions within a colorimetric assay?
What is the consequence of not achieving equilibrium in early-stage reactions within a colorimetric assay?
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Which equation is used to calculate the unknown protein concentration in the Bradford Assay?
Which equation is used to calculate the unknown protein concentration in the Bradford Assay?
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Which of the following describes qualitative colorimetric assays?
Which of the following describes qualitative colorimetric assays?
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Study Notes
Colorimetric Assays
- Assays use reagents that change color in the presence of an analyte (chemicals like enzymes, proteins, antibodies or hormones)
- Binary Assays: Provide positive/negative results (e.g., pregnancy tests)
- Qualitative Assays: Estimate analyte concentration using color charts (like pH litmus paper or aquarium test strips)
- Quantitative Assays: Measure photon absorption using a spectrophotometer at specific wavelengths.
- Quantitative Assays correlate absorbance with analyte concentration using a standard curve.
The Bradford Assay
- A colorimetric assay for protein detection.
- Coomassie blue dye changes from orange-red to blue when bound to amino acids.
- Maximum absorbance shifts from 465 nm to 595 nm.
- The assay completes in less than 5 minutes.
- Quantitative Analysis: Uses a linear standard curve to define protein concentration.
- Protein concentration (µg/mL) calculation: 100.46 * absorbance at 565 nm
Beer-Lambert Law
- Describes the relationship between absorbance (A), molar absorptivity (ε), path length (b), and molar concentration (c): A = εbc
Standard Curve Analysis
- Essential for identifying analyte concentration relationships.
- Reliable within the linear range (a high R² value, ideally ≥ 0.95).
- Plot absorbance versus concentration for known samples.
- Exclude outliers outside the linear range.
- The equation of the linear trendline shows the relationship between absorbance and concentration.
- High R²: Indicates a good fit and accurate relationship.
- Low R²: Signals significant deviations, suggesting unreliable data.
Dilution of Solutions
- Formula: M1V1 = M2V2
- M1 = initial concentration, V1 = initial volume
- M2 = final concentration, V2 = final total volume
- Example: Diluting a 1 mg/mL protein sample in a 1:5 ratio (100 µL + 400 µL PBS) results in a 0.2 mg/mL final concentration.
Molar Concentration
- Molar concentration (M) = (Mass concentration, g/L) / Molecular weight (g/mol)
Errors in Measurements
- Sources: Inconsistent sample preparation, colorimeter calibration inaccuracies, reactions not reaching equilibrium, contamination or human error.
- Improvement Tips: Instrument calibration before use, consistent sample handling, allowing reactions to reach equilibrium.
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Description
This quiz covers colorimetric assays, including the Bradford assay for protein detection. It explores binary, qualitative, and quantitative assay methods, as well as the Beer-Lambert law and its application in measuring analyte concentrations. Test your knowledge on these essential biochemical techniques!
