Biochemical separation

This week I'm doing something a little bit different, to show you precisely what goes on in a first-year vet school lab. Last week we had two labs, one on enzyme kinetics, and one on biochemical separation methods. So in this week's blog post, I thought I'd show you how we worked, what equipment we used, and some pictures from the lab, and us working. So without further ado, here are the two first tasks from out biochemical separation lab:

For our first lab, we were given five tasks, all related to biochemical separation. Here we used spectrophotometry, gel-filtration chromatography and gel electrophoresis to determine what chemical components were in different solutes, as well as to determine what the molecule weight was.

Two of the five tasks were conducted in the lab, and for those we used some of the equipment from the image below:

Task 1: Determining the best wavelength at which to measure absorbance

Equipment:

  • Cuvettes
  • Automatic pipettes
  • Pipette caps
  • Spectrophotometer
  • CoCl2 (Cobalt chloride)
  • H2O (as a blind test)

The way a spectrophotometer works is by measuring the light absorption of different biomolecules at different wavelengths. This is done by shining a light source through a monochromatic that makes the broad-spectrum light the same wavelength. It then uses what is known as the “Lambert-Beer law” to calculate the fractional relationship between the light that was shone onto the cuvette, also known as the incident light (I0), and the light that comes out on the other side of the cuvette, namely the transmitted light (I). By doing this we can detect and identify molecules, and measure their concentration in a given solution.

In the first task, we set the spectrophotometer to 460, using the water as a blind test to reset the machine, and measured how much light CoCl2 absorbed, increasing the wavelength by 10 nanometres each time we measured, all the way up to 600 nm. We found that the solute had the highest absorbance at about 510 nm, thereby determining that this would be the best wavelength at which to continue our quantitative measurements.

Task 2: Using gel filtration to separate a protein from a salt

  • Cuvettes
  • Automatic pipettes
  • Pipette caps
  • Spectrophotometer
  • Cylindrical column filled with a permeable solid matrix (Sephadex G-25, through which we ran the solutions)
  • BD (Blue dextran)
  • BSA (bovine serum albumin)
  • CoCl2 (Cobalt chloride)
  • An unknown test solution
  • Distilled H2O (used here as a buffer)

For the second task, we used a method called “gel-filtration chromatography” to separate proteins according to their size. This is done by sifting the solute through a porous gel matrix, in this case “Sephadex G-25”, where protein molecules that are small enough will enter the beads in the matrix. In a G-25 matrix, that would only include molecules that have a molecule weight lighter than 5 kilo Daltons. After running the unknown test solution through the matrix, we used water to wash out the pores, bringing out different molecules at different stages according to their weight. The heavier the protein molecules were, the sooner they would be washed out of the gel-filtration column. We caught the solution as it was dripping out of the cylindrical column, changing cuvettes every 18th drip. What we noticed was that between the 12 columns, there were many different colours, and different gradients of the same colours. We then used the spectrophotometer at the different optimal wavelengths for BD, BSA and CoCl2, and decided which cuvettes contained which solutes, which again gave us an idea of what the unknown test solution contained. We ended up with a graph that looked like this:

We also measured four cuvettes containing BSA, CoCl2, BD and the unfiltered unknown solution, and got these results:

Note: we added 1 ml of BSA to 17 new cuvettes after running the first samples through the spectrophotometer, and added 20 µl of the original 17 samples to get the results for the 595 nm spectrophotometry.

The final three task were mostly calculation based, so I'll spare you the not-so-interesting equations. I got a request over at Instagram (click here to follow me) to include a subscription link, so if you're interested in subscribing to the blog you can go over to bloglovin' and follow me there, or you can click the RSS-feed here:

Other than that, I hope you're all having a lovely Sunday!