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. Dounce Homogenizer | ||
| 2. Freeze-thaw | ||
| 3. Mortar & Pestle | ||
| 4. Microfluidizer |
Drag one of the choices below:
- A. This technique uses extremely cold temperatures and grinding to pulverize cells.
- B. A spinning handheld device smashes cells in a tube.
- C. This is laborious manual work that can take several minutes.
- D. A high-pressure pump pushes cells through a Z-shaped interaction chamber.
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. gel filtration column (GFC) | ||
| 2. hydrophobic interaction column (HIC) | ||
| 3. affinity column (AC) | ||
| 4. ion exchange column (IEX) |
Drag one of the choices below:
- A. for one type, high salt concentration enhances the interaction, whereas low salt concentrations weaken it
- B. relies on the specific and reversible binding of a protein to a matrix-bound ligand
- C. relies on charge-charge interactions between the proteins in your sample and the charges on the particle resin
- D. size separation in a column filled with porous beads
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 proteins | ||
| 2. Both protein and nucleotide | ||
| 3. Only nucleotides |
Drag one of the choices below:
- A. PAGE
- B. two-dimensional gels
- C. agarose gels
Cell Disruption Techniques from Descriptions (MC)
Click to show Cell Disruption Techniques from Descriptions (MC) example problem
Which one of the following cell disruption techniques correspond to the description 'Manual grinding of cells that can take several minutes."'.
Types of Column Chromatography Based on Descriptions
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Which one of the following types of column chromatography correspond to the description 'separates proteins according to differences in their surface hydrophobicity'.
Matching Macromolecule Types to Gel Electrophoresis Processes
Click to show Matching Macromolecule Types to Gel Electrophoresis Processes example problem
Which one of the following types of macromolecules correspond to the gel component or process 'native gels'.
Protein Net Charge at a Given pH
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Isoelectric Point Problem
| Protein Name | isoelectric point (pI) | molecular weight |
|---|---|---|
| G3P Dehydrogenase (GDH) | 8.3 | 36.0 |
The protein in the table (above) is placed in a buffer solution with a pH of 9.5.
What is the correct net charge on the GDH protein at pH of 9.5
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 |
|---|---|---|
| Casein (Cas) | 4.9 | 24.0 |
| Enolase (Eno) | 8.4 | 42.5 |
Both protein samples are placed into a gel with a constant pH of 6.5. The gel is then placed into an electric field.
In which direction will each protein in the table migrate at pH 6.5?
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 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) |
|---|---|---|
| Cytochrome c (Cyt) | 13.0 | 3.45 |
| Myelin Basic Protein (MBP) | 18.0 | 3.19 |
| Agglutinin (Agg) | 22.0 | 3.03 |
| Elastase II (Ela) | 26.5 | 2.88 |
| Pepsin (Pep) | 34.5 | 2.67 |
| Ovalbumin (Ova) | 45.0 | 2.45 |
| γ-Globulin (Glob) | 57.0 | 2.27 |
| 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 7% of the correct number to be correct.