User:Zachary I. Mendel/Notebook/Zacks 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
  3. 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
    2. Add 200μL of the Bio-Rad Protein Assay reagent
    3. Add the correct amount of buffer such that the final volume is 1mL
    4. Close the tubes and vortex them.
    5. Let them sit for 5 minutes
  4. Take a UV-Vis (no less than 1 hour after they were produced).
    1. Use the plastic cuvettes.
  5. 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)
  6. After you have finished one set, repeat the process (make new samples and new measurements)

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

  1. Make a calibration curve.
  2. Determine if you need to redo any data or sample prep.

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

Notes

Summary:

Today's experiment certainly did not go as planned. We took absorbance readings for three trials of the adenosine deaminase and did not get good readings on any of the trials. Dr. Hartings has instructed us to repeat the experiment tomorrow the 11th using 2ml volumes as opposed to 1ml readings. He hopes this will correct the problem.

It is important to note that when we were making our initial stock solution of adenosine deaminase, we were only able to obtain 3.67ug of protein as opposed to the 10ug that we were hoping to obtain.

Adenosine Deaminase Table


Below are the absorbance spectra for trial 1 and trial 2.

ADA Trial 1 Trail 1 ADA.jpg


ADA Trial 2 ADA Trial 2.jpg


Here is the data table for the making of the gold standards solutions

Gold Nanoparticles Standard.png