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 
Cell Disruption Techniques from 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. French Press | ||
| 2. Freeze-thaw | ||
| 3. Enzymatic Method | ||
| 4. Mortar & Pestle |
Drag one of the choices below:
- A. Extreme temperature variations cause cell wall rupture.
- B. Digestion of the cell wall by chemical reactions.
- C. Cells are forced through a tiny hole by a high-pressure hydraulic piston.
- D. Manual grinding of cells that can take several minutes."
Column Chromatography Types from 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. ion exchange column (IEX) | ||
| 2. affinity column (AC) | ||
| 3. gel filtration column (GFC) | ||
| 4. hydrophobic interaction column (HIC) |
Drag one of the choices below:
- A. separates by surface hydrophobicity, particle binding is controlled by salt concentration and/or organic solvents
- B. ligand-attached beads bind directly to the protein of interest
- C. separation method essentially based on the net charge of the protein
- D. ability to separate protein samples by size using porous particles
Protein and Nucleic Acid Gel Electrophoresis Components
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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. β-mercaptoethanol (βME)
- B. SYBR safe stain
- C. electrical fields
Cell Disruption Techniques from Descriptions
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Which one of the following cell disruption techniques correspond to the description 'Large numbers of samples are disrupted simultaneously using a bead-beating machine.'.
Column Chromatography Types from Descriptions
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Which one of the following types of column chromatography correspond to the description 'works using resin-attached ligands to grab hold of the corresponding proteins'.
Protein and Nucleic Acid Gel Electrophoresis Components
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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 |
|---|---|---|
| Tropomyosin (Trop) | 5.1 | 35.0 |
The protein in the table (above) is placed in a buffer solution with a pH of 6.5.
What is the correct net charge on the Trop protein at pH of 6.5
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 |
|---|---|---|
| Fibrinogen (Fib) | 5.8 | 63.5 |
| Immunoglobulin (IgG) | 7.3 | 145.0 |
Both protein samples are placed into a gel with a constant pH of 8.5. The gel is then placed into an electric field.
In which direction will each protein in the table migrate at pH 8.5?
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 band labeled Protein X17.
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:
Which protein (name and molecular weight) best matches Protein X17?
Use the ladder to estimate the band size. You do not need outside knowledge about the proteins.
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) |
|---|---|---|
| Hemoglobin (Hem) | 16.7 | 3.25 |
| Succinate Ligase (SL) | 20.9 | 3.07 |
| Urease A (Ure) | 26.5 | 2.88 |
| Horseradish peroxidase (HP) | 34.0 | 2.68 |
| Enolase (Eno) | 42.5 | 2.50 |
| Serum Albumin (Alb) | 66.2 | 2.15 |
| Unknown | ? | 2.77 |
Estimate the molecular weight of the unknown protein by comparing its gel migration distance with those of the standards.
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
H3N+
COOH
NH3+
CH3
|
→ |
State 2
H3N+
COO−
NH3+
CH3
|
→ |
State 3
H3N+
COO−
NH2
CH3
|
→ |
State 4
H2N
COO−
NH2
CH3
|
|
|
||||||||||||||||||||||||||||||||||||||||
Given pKa values: pKa1 = 2.1, pKaR = 6.5, pKa2 = 9.7.
The isoelectric point (pI) is closest to which value?