Activity 3: Guide
Module Overview
📚Introduction
The extracted proteome, or total proteins expressed, from a biological sample (e.g., plant samples) contains proteins of different properties, such as size and charge. These differences can be useful in separating and differentiating proteins present in the mixture. A good method for protein separation and proteome characterization is polyacrylamide gel electrophoresis (PAGE). In this experiment, extracted total protein will be characterized using discontinuous Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis (SDS-PAGE) in Laemmli buffer system.
SDS-PAGE is commonly used in research laboratories to separate pure protein extracts according to their molecular weight. SDS, an anionic detergent allows denaturation of proteins into linear structures and gives denatured proteins an overall negative charge, thus canceling out any intrinsic charges that are present in the undenatured form. The polyacrylamide gel serves as a sieving matrix through which proteins, mobilized by electric current, pass through. Smaller proteins tend to migrate faster than larger ones and seen at the far points from the wells.
A standard or molecular weight ladder is used in order to determine the approximate sizes of the separated proteins. After electrophoresis, the protein bands are visualized using staining methods. A commonly used stain is Coomassie blue. It is cheap and easy to perform, and is also compatible with mass spectrometry and other downstream assays. However, Coomassie blue staining has low sensitivity; proteins should be at least 1 mg for them to be visualized. For detection of low-concentration proteins,silver staining is a viable alternative. Although it requires more time, steps, and reagents, silver staining allows detection of proteins with as low as 1 ng concentration. However, reagents used in this method are toxic, which means extra care is needed during staining.
In this activity, you should be able to:- Explain the principles behind discontinuous SDS-PAGE
- Analyze and interpret protein profiles
⚙️Module Tasks
- Read the guide.
- Watch the SDS-PAGE demo video.
- Perform the simulation for SDS-PAGE with varying conditions.
- Interpolate MW from SDS-PAGE profile.
- Answer the problem set associated with this activity.
⏰Time Allotment: 2 - 4.5 hrs
Suggested Time Frame per Task:
| Task | Allotted Time | In Pomodoro Terms (🍅) |
|---|---|---|
| Overview | 15 min | <1 |
| Watch the video | 15 min | <1 |
| Perform the simulation and compute for MW | 25-50 min | 1-2 |
| Answer the probset (spaced in a week) | 1-3 hrs | 2-8 |