5: Protein Purification
Isolation and analysis methods, chromatography, electrophoresis.
LibreTexts reference: Protein Purification
Matching Cell Disruption Techniques to Descriptions
Click to show Matching Cell Disruption Techniques to Descriptions example problem
Match each of the following cell disruption techniques with their corresponding descriptions.
Note: Each choice will be used exactly once.
| Your Choice | Prompt | |
|---|---|---|
| 1. French Press | ||
| 2. Mortar & Pestle | ||
| 3. High-throughput Homogenizer | ||
| 4. Sonication |
Drag one of the choices below:
- A. Cells are pushed through a small hole using high pressure from a hydraulic piston.
- B. High-frequency sound waves produce tiny bubbles that explode, producing a local shockwave.
- C. An expensive bead-beating machine can process a large number of samples simultaneously in a short time.
- D. Manual grinding of cells that can take several minutes."
Matching Column Chromatography Types to Descriptions
Click to show Matching Column Chromatography Types to Descriptions example problem
Match each of the following types of column chromatography with their corresponding descriptions.
Note: Each choice will be used exactly once.
| Your Choice | Prompt | |
|---|---|---|
| 1. ion exchange column (IEX) | ||
| 2. size-exclusion column (SEC) | ||
| 3. reverse phase column (RPC) | ||
| 4. affinity column (AC) |
Drag one of the choices below:
- A. only liquid chromatography method where molecules do NOT bind to the chromatography particles
- B. technique based on the ability to separate proteins based on relative hydrophobic differences
- C. relies on the specific and reversible binding of a protein to a matrix-bound ligand
- D. separates proteins with differences in surface charge
Matching Macromolecule Types to Gel Components or Processes
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Match each of the following types of macromolecules with their corresponding gel components or processes.
Note: Each choice will be used exactly once.
| Your Choice | Prompt | |
|---|---|---|
| 1. Only nucleotides | ||
| 2. Both protein and nucleotide | ||
| 3. Only proteins |
Drag one of the choices below:
- A. sodium dodecyl sulfate (SDS)
- B. polyacrylamide gel electrophoresis
- C. agarose gels
Cell Disruption Techniques from Descriptions (MC)
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Which one of the following cell disruption techniques correspond to the description 'Chemical reactions digest the cell wall.'.
Types of Column Chromatography Based on Descriptions
Click to show Types of Column Chromatography Based on Descriptions example problem
Which one of the following types of column chromatography correspond to the description 'above its isoelectric point (pI), a protein will bind to a positively charged anion exchanger'.
Matching Macromolecule Types to Gel Electrophoresis Processes
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Which one of the following types of macromolecules correspond to the gel component or process 'β-mercaptoethanol (βME)'.
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 |
|---|---|---|
| Cobra Venom Factor (CVF) | 5.2 | 149.0 |
The protein in the table (above) is placed in a buffer solution with a pH of 4.0.
What is the correct net charge on the CVF protein at pH of 4.0
Protein Migration Direction from Isoelectric Point
Click to show Protein Migration Direction from Isoelectric Point 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 |
|---|---|---|
| Pyruvate Kinase (PK) | 5.6 | 60.0 |
| Aprotinin (Apr) | 9.2 | 6.5 |
Both protein samples are placed into a gel with a constant pH of 10.5. The gel is then placed into an electric field.
In which direction will each protein in the table migrate at pH 10.5?
Calculating Molecular Weight from SDS-PAGE Ladder
Click to show Calculating Molecular Weight from 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 long a time (some small bands may run off the bottom).
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 |
| Lysozyme (Lys) | 14.4 | 3.37 |
| Succinate Ligase (SL) | 20.9 | 3.07 |
| Chymotrypsin (Chy) | 25.0 | 2.92 |
| Pepsin (Pep) | 34.5 | 2.67 |
| Enolase (Eno) | 42.5 | 2.50 |
| Aldehyde Dehydrogenase (Alde) | 53.0 | 2.32 |
| Unknown | ? | 2.77 |
Estimate the molecular weight of the unknown protein by comparing its gel migration distance with those of the standards.
Note: answers need to be within 8% of the correct number to be correct.