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: Analyzing samples by SDS PAGE electrophoresis

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

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

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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

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