Quantitative Detection of Protein (SpectroVis Plus)

Experimental Background

Bovine Serum Albumin (BSA) is a protein that circulates in the blood of cows. Purified BSA can be used with Biuret solution in serial dilutions to generate a Standard Curve. The standard curve will illustrate the relationship between concentration (the dependent variable) and absorbance at 540 nm (the independent variable).  We can then use this curve to estimate the concentration of unkown samples.

  1. On a graph, do you remember which axis is the dependent and which is the independent variable?
  2. In the table below, can you identify which samples are the negative controls and which are the positive controls?
  3. What is the prediction of the absorbance or color intensity of the different tubes?

Dilute BSA Standards

  1. Label 9 tubes 1-9
  2. Combine the components of the table below to generate appropriate concentration of solutions

Dilution table

  1. The instructor will begin to set-up the units for distribution
    1. Connect SpectroVis Plus to LabQuest2
    2. Power on
    3. Select LabQuest App
    4. On the “Meter” tab, tap on Mode
      1. Change the mode to “Events with Entry
      2. Enter the Name: Concentration
      3. Enter Units: mg/ml
      4. Select “OK”
      5. If message appears about saving run, choose Discard
      6. Tap on the red area showing Absorbance to trigger menu items
        1. Select “Change Wavelength
        2. Enter 540
      7. Tap on the red area showing Absorbance to trigger menu items
        1. Select “Calibrate”
        2. Select “Warm Up”
        3. Select Finish Calibration
  2. Place tube 1 (0 mg/ml) into a cuvette for measuring absorbance (A) in the SpectroVis Plus.
    • tap on the file cabinet icon to store this data
  3. Sequentially read each sample at the stored wavelength (A540nm)and record values in table below

Data recording

  1. Plot each BSA dilution in plot.ly as a scatterplot (Log-on using Facebook/Google/Twitter credentials for free)
  2. Generate best-fit line for these standards with the equation of the line
  3. Use the equation of the line to estimate the concentration of the unknown sample.

Curve Fitting

Run the simulation below to understand how you can use the standard dilution series to estimate your sample concentrations.

Curve Fitting
Click on image above to begin simulation on curve fitting

LabQuest2 and SpectroVis Tutorial

Scatterplot Tutorial

You can watch this tutorial at 1.25X and pause when needed.


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