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Protocol and Writeup for Week 2 (Biology 4412 Spring 2021): 30 points

Part I: Analyzing samples by SDS PAGE electrophoresis—13 pts

This week you will analyze the samples you prepared last week, and use SDS-PAGE to separate proteins by mass.

The gel is made of polyacrylamide and contains the detergent SDS. The SDS denatures the proteins and coats them, providing a negative charge around each protein. This allows us to use electrophoresis to separate proteins by mass (NOT size). Heavier proteins migrate slower (and thus are near the top) and lighter proteins migrate more quickly (and thus are near the bottom). You will note that the gel is vertical, which is different than agarose gel electrophoresis. We will use a running buffer (typically 1X Tris-Glycine) to fill the tank of the electrophoresis chamber.

The samples from last week will be prepared by adding Laemmli Sample Buffer and boiling them. You will then load the sample on the gel, and then the gel will be electophoresed. We will use a protein “ladder” that will allow us to determine the approximate mass of the proteins we separate. Once the samples are loaded, the gel will run and then will be stained with a dye called Coommassie Blue (the same dye as in the Bradford assay) to stain proteins. This will happen after your lab time, so the pictures of the final stained gel will be posted to OneNote.

Preparing the samples for electrophoresis

1. You will receive your protein sample that you extracted last week.

2. Transfer 50 uL of your sample to a new 1.5 mL microfuge tube.

3. Then, add 50 uL of the 2X Laemmli Sample buffer to your sample. This buffer contains SDS and the reducing agent B-mercaptoethanol which will fully denature the proteins prior to loading on the gel. The buffer also contains a dye which will aid in loading the gel in the next part of this activity.

4. Put a cap on your tube (to prevent the tube from opening) and place the tube into the 95C heat block for 5 minutes.

5. Centrifuge the tube for 1 minute at 12,000 rpm. When you remove the tubes, you may see a small precipitate (pellet) at the bottom of the tube. The proteins you want are in the supernatant of the tube.

6. Transfer the supernatant to a new 1. 5 mL microfuge tube.

Loading the samples on the gel

Each gel will be shared per lab session, and each one of you will load your sample into the gel. The instructor will load a protein size standard on the gel, and give you the profile of these sizes.

Before loading samples, we wil carefully remove the comb from the gel by pulling it straight up. You will see the wells that were formed by the comb, and they should fill with the buffer that is present in the apparatus.

The instructor will load the size standard sample so that you all can see how to load samples, and you can refer to the directions below as well.

For each sample:

1. Remove 50 ul of the sample. Some of the samples may be very viscous ("gooey") because of extracted DNA.

2. Place the pipet tip above the sample well. You will have to angle the tip of the pipet so that it is almost between the glass plates, but you do not want to jam the pipet between the plates.

3. Eject all the sample from the pipet. You should see it fill up the well. Do NOT release the plunger before all the sample is ejected.

Once all the samples are loaded, before moving to step 4, each one of you should record the ORDER of the samples loaded on the gel.

4. Once the samples are loaded, carefully attach the electrode top to the chamber. The apparatus is set up so that the red electrode fits into the red slot, and the black electrode fits into the black slot. Red is positive input, black is negative input.

5. Connect the leads to the power supply. Set the power supply to deliver 150 Volts. You should see the presence of bubbles on the electrodes, indicating that current is flowing.

6. Allow the gel to run for approximately one hour (until the blue dye is about 1-2 cm from the bottom of the plate).

The gel will be stained with Coomassie Blue by your instructor, and the photos of the gels will be posted to either OneNote or Blackboard for you to use in the analysis.

Part II: Practice with SDS PAGE interpretation: Before you do your analysis, I would like you to practice with the following example. –5 pts

(5 pts) The figure below shows a sample SDS-PAGE gel that has been stained with the dye Coomassie Blue. The gel shows mass standards (in kDa) on the left hand side. There are 9 samples (lanes) shown on the gel (not counting the mass standards, which is not numbered)

For each lane, describe/list the mass of the most abundant protein(s) in each lane. (I’m asking for the mass of the bright blue bands in each lane, and some have more than one)

Part III: Analysis of your gel—12 pts

(2) 1. In the space below, copy and paste the photo of your groups’ gel.

(6) 2. Can you identify differences in the pattern and abundance of the proteins from control and treated samples? Explain your answer with specific reference to the gel that you posted in question 1.

(4) 3. Based on your results, can you make any conclusions on how induction of oxidative stress by hydrogen peroxide affected protein translation? (this is based on your results, not what the “right” answer is).