User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/09/10

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Objective

The primary way of determining protein concentration is through a measurement of the protein's UV-Vis spectrum and using its molar absorptivity at 280nm to calculate concentration. For low concentrations of proteins, UV-Vis of just the protein is often not sensitive enough to accurately measure concentration. During the semester, we may need to measure protein concentrations that are very low. One chemical tool that we can use to do this is called the Bradford Assay. The Bradford Assay makes use of the Coomassie Blue dye, which binds to proteins. Upon binding to a protein, this dye undergoes a change in its absorption features. (No protein: peak at 460. Protein: peak at around 600). We will be making calibration curves (using the Bradford Assay) for the different proteins we'll be using throughout the semester.

Description

The basic protocol that we will be using for this procedure can be found here. (*Note: use section 2.3, page 5)

  1. Make a stock solution that is roughly 10mg protein in 2mL of buffer. (there are 2-2mL volumetric flasks that you will have to share)
  2. Calculate what your actual concentration
        - We used pepsin, and we calculated that our actual concentration is 5.6 for trial 1, and 5.9 for trial 2
  1. Make 6 standard solutions (1mL each) between 1μg/mL and 10μg/mL
    1. Determine the appropriate volume of your stock to use (for the proper final concentration in 1mL) and add that volume to an eppendorf tube

"Table 1. Concentration and volumes used for the 6 solutions made, Trial 1"

Comcentration(M) volume Concentration pepsin volume (uL) volume buffer
0.5 1000 5.6 89.2857143 910.714286
1 1000 5.6 178.571429 821.428571
1.5 1000 5.6 267.857143 732.142857
2 1000 5.6 357.142857 642.857143
2.5 1000 5.6 446.428571 553.571429
3 1000 5.6 535.714286 464.285714
0.25 1000 5.6 44.6428571 955.357143
0.1 1000 5.6 17.8571429 982.142857


"Table 2. Concentration and volumes used for the 6 solutions made, Trial 2"

Comcentration(M) volume Concentration pepsin volume (uL) volume buffer
0.5 1000 5.9 84.7457627 915.254237
1 1000 5.9 169.491525 830.508475
1.5 1000 5.9 254.237288 745.762712
2 1000 5.9 338.983051 661.016949
2.5 1000 5.9 423.728814 576.271186
3 1000 5.9 508.474576 491.525424
0.25 1000 5.9 42.3728814 957.627119
0.1 1000 5.9 16.9491525 983.050847


    1. Add 200μL of the Bio-Rad Protein Assay reagent
    2. Add the correct amount of buffer such that the final volume is 1mL
    3. Close the tubes and vortex them.
    4. Let them sit for 5 minutes
  1. Take a UV-Vis (no less than 1 hour after they were produced).
    1. Use the plastic cuvettes.
  2. Make 2 blanks as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum. (400nm-800nm)
    1. We will be using one of the blanks as a reference for each spectrum that we take. I'll show you where to place this cuvette for each spectrum you collect)
  3. After you have finished one set, repeat the process (make new samples and new measurements)

"Figure 1. Absorbance vs wavelength in Trial 1 of pepsin"

Absorbance vs Wavelength of Trial 1.png

"Figure 2. Absorbance vs wavelength in Trial 2 of pepsin"

Absorbance vs wavelength trial 2.png

- Note: This is where coordination is a good thing. Take 1 spectrum at a time. Let other people go.

  1. Make a calibration curve.

"Figure 3. Calibration curve of Trial 1 pepsin"

Calibration curve trial 1 pepsin.png

"Figure 4. Calibration curve of trial 2 pepsin"

Calibration curve trial 2 pepsin.png

"Figure 5. Calibration curve of both trials"

Calibration curve for both trials.png

  1. Determine if you need to redo any data or sample prep.
   - We had to redo two samples. We discarded the data of sample 2.5 and 3, because their absorbance was over 1, and we used 0.25 and 0.1 for our calibration curves

Note - Solutions from today containing the stain will go into a waste container in the hood.

We are also going to make Atomic Absorption standards for tomorrow!

Using the gold AA/ICPMS standard solution make 5 new solutions (Note: Use water - NOT BUFFER - to make these solutions)

  1. 25 ug/mL
  2. 20 ug/mL
  3. 15 ug/mL
  4. 10 ug/mL
  5. 5 ug/mL

Data

Making buffer: 1L 50mM Tris 50mM NaCl pH 7.5

Should mass out:

  1. 6.057g Tris
  2. 2.922g NaCl

Actually measured:

  1. 6.0596g Tris
  2. 2.9525g NaCl

Gold solution for group: HAT to make nano particles

  1. Gold solution (HAuCl4·3H2O) 0.0050g in 0.0500mL water → 0.25mM