5: Protein Purification
Students choose purification methods for proteins based on size, charge, and binding affinity, and interpret results from gel filtration, ion exchange, SDS-PAGE, and isoelectric focusing.
LibreTexts reference: Unit 1, Chapter 5: Protein Purification 
Matching Cell Disruption Techniques to Descriptions
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Match each of the following cell disruption techniques with their corresponding descriptions.
Note: Each choice will be used exactly once.
| Your Choice | Prompt | |
|---|---|---|
| 1. High-throughput Homogenizer | ||
| 2. French Press | ||
| 3. Mechanical Homogenizer | ||
| 4. Sonication |
Drag one of the choices below:
- A. Ultrasonic sound energy is used to cause air bubbles in a liquid to implode, damaging the cells.
- B. Large numbers of samples are disrupted simultaneously using a bead-beating machine.
- C. Shearing forces from spinning blades break cells apart.
- D. High-pressure hydraulic piston forces cells through a small hole.
Matching Column Chromatography Types to Descriptions
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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. reverse phase column (RPC) | ||
| 2. ion exchange column (IEX) | ||
| 3. affinity column (AC) | ||
| 4. gel filtration column (GFC) |
Drag one of the choices below:
- A. below its isoelectric point (pI), a protein will bind to a negatively charged cation exchanger
- B. size separation in a column filled with porous beads
- C. separates by surface hydrophobicity, particle binding is controlled by salt concentration and/or organic solvents
- D. a specific ligand is attached to the particle resin in the column
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. Only proteins | ||
| 3. Both protein and nucleotide |
Drag one of the choices below:
- A. gel red stain
- B. stain / destaining process
- C. polyacrylamide gel electrophoresis
Cell Disruption Techniques from Descriptions
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Which one of the following cell disruption techniques correspond to the description 'This technique uses the forces of ultrasonic waves to rupture cells.'.
Column Chromatography Types from Descriptions
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Which one of the following types of column chromatography correspond to the description 'relies on charge-charge interactions between the proteins in your sample and the charges on the particle resin'.
Macromolecule Types from Gel Electrophoresis Descriptions
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Which one of the following types of macromolecules correspond to the gel component or process 'heating to almost boiling (95°C)'.
Protein Net Charge at a Given pH
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Isoelectric Point Problem
| Protein Name | isoelectric point (pI) | molecular weight |
|---|---|---|
| Fumerase (Fum) | 7.6 | 48.5 |
The protein in the table (above) is placed in a buffer solution with a pH of 9.0.
What is the correct net charge on the Fum protein at pH of 9.0
Protein Migration Direction in Isoelectric Focusing
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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 |
|---|---|---|
| Cobra Venom Factor (CVF) | 5.2 | 149.0 |
| Cytochrome c (Cyt) | 10.2 | 13.0 |
Both protein samples are placed into a gel with a constant pH of 7.5. The gel is then placed into an electric field.
In which direction will each protein in the table migrate at pH 7.5?
Protein Molecular Weight from an SDS-PAGE Ladder
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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 a typical amount of time.
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 10% of the correct number to be correct.
Protein Molecular Weight from SDS-PAGE Migration
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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) |
|---|---|---|
| Lysozyme (Lys) | 14.4 | 3.37 |
| Myoglobin (Myo) | 18.0 | 3.19 |
| Trypsin (Tryp) | 23.5 | 2.97 |
| Prostate-Specific Antigen (PSA) | 30.0 | 2.78 |
| G3P Dehydrogenase (GDH) | 36.0 | 2.63 |
| Aldolase (Aldo) | 47.5 | 2.41 |
| α-Amylase (Amy) | 61.0 | 2.21 |
| Unknown | ? | 3.30 |
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 6% of the correct number to be correct.
Isoelectric Point from pKa Values
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Isoelectric Point from Titration Curve
Below are four protonation states of a hypothetical molecule, shown in order as the solution is titrated from low pH to high pH.
State 1
COOH
CH3
H3N+
COOH
|
→ |
State 2
COOH
CH3
H3N+
COO−
|
→ |
State 3
COO−
CH3
H3N+
COO−
|
→ |
State 4
COO−
CH3
H2N
COO−
|
|
|
||||||||||||||||||||||||||||||||||||||||
Given pKa values: pKa1 = 2.5, pKaR = 4.6, pKa2 = 10.2.
The isoelectric point (pI) is closest to which value?