3: Protein Biotechnology
Students examine proteomics, western blotting, mass spectrometry, phage display, ELISA, recombinant expression systems, directed evolution, AlphaFold, cryo-EM, and de novo protein design.
Protein Net Charge at a Given pH
Click to show Protein Net Charge at a Given pH example problem
Isoelectric Point Problem
| Protein Name | isoelectric point (pI) | molecular weight |
|---|---|---|
| Immunoglobulin (IgG) | 7.3 | 145.0 |
The protein in the table (above) is placed in a buffer solution with a pH of 8.5.
What is the correct net charge on the IgG protein at pH of 8.5
Protein Migration Direction in Isoelectric Focusing
Click to show Protein Migration Direction in Isoelectric Focusing example problem
Isoelectric Point Problem
A protein's isoelectric point (pI) is the pH at which it carries no net charge. When placed in a pH environment different from its pI, the protein will acquire a net charge and migrate in an electric field accordingly.
A mixture of two proteins are to be separated by isoelectric focusing.
| Protein Name |
Isoelectric Point (pI) |
Molecular Weight |
|---|---|---|
| γ-Globulin (Glob) | 6.6 | 57.0 |
| Ribonuclease A (RibA) | 9.3 | 13.7 |
Both protein samples are placed into a gel with a constant pH of 8.0. The gel is then placed into an electric field.
In which direction will each protein in the table migrate at pH 8.0?
Protein Molecular Weight from an SDS-PAGE Ladder
Click to show Protein Molecular Weight from an SDS-PAGE Ladder example problem
Below is a simulated SDS–PAGE gel.
Lane 1 contains a Kaleidoscope-style pre-stained protein ladder. Lane 2 contains a single protein band.
The gel was run for too short a time (bands are compressed near the top).
Standard ladder reference (kDa):
| – 250 | ||||
| – 150 | ||||
| – 100 | ||||
| – 75 | ||||
| – 50 | ||||
| – 37 | ||||
| – 25 | ||||
| – 20 | ||||
| – 15 | ||||
| – 10 | ||||
Gel results:
What is the molecular weight (kDa) of the band in lane 2?
Assume ln(MW) is approximately linear with migration distance.
Note: answers need to be within 11% of the correct number to be correct.
Protein Molecular Weight from SDS-PAGE Migration
Click to show Protein Molecular Weight from SDS-PAGE Migration example problem
Gel Migration Problem
In this task, data from an SDS-PAGE experiment, where proteins are separated based on molecular weight, is provided. The gel results table below shows some standard proteins with known molecular weights and one unknown protein.
| Protein Name | Molecular Weight (kDa) |
Migration Distance (cm) |
|---|---|---|
| Lactalbumin (Lac) | 13.0 | 3.45 |
| Avidin (Av) | 16.9 | 3.24 |
| Serine Protease (Ser) | 22.0 | 3.03 |
| Prostate-Specific Antigen (PSA) | 30.0 | 2.78 |
| Lipase (Lip) | 40.0 | 2.55 |
| Luciferase (Luc) | 50.0 | 2.37 |
| Catalase (Cat) | 65.5 | 2.15 |
| Unknown | ? | 2.23 |
Estimate the molecular weight of the unknown protein by comparing its gel migration distance with those of the standards.