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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 LibreTexts

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
Drop Your Choice Here 1. French Press
Drop Your Choice Here 2. High-throughput Homogenizer
Drop Your Choice Here 3. Mortar & Pestle
Drop Your Choice Here 4. Mechanical Homogenizer

Drag one of the choices below:

  • A. Drill-like device with spinning rotor blades to shear cells apart.
  • B. Samples are ground by hand to break apart into small pieces.
  • C. Large numbers of samples are disrupted simultaneously using a bead-beating machine.
  • D. A hydraulic piston within a cylinder forces cells through a small hole using high pressure.
 

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
Drop Your Choice Here 1. ion exchange column (IEX)
Drop Your Choice Here 2. affinity column (AC)
Drop Your Choice Here 3. hydrophobic interaction column (HIC)
Drop Your Choice Here 4. size-exclusion column (SEC)

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. charged proteins interact with an oppositely charged chromatography particle resin
  • 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
Drop Your Choice Here 1. Only proteins
Drop Your Choice Here 2. Both protein and nucleotide
Drop Your Choice Here 3. Only nucleotides

Drag one of the choices below:

  • A. acrylamide
  • B. Coomassie blue stain
  • C. ultraviolet light (UV) visualization
 

Cell Disruption Techniques from Descriptions (MC)

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Which one of the following cell disruption techniques correspond to the description 'The cells are disrupted by hand grinding them.'.

 

Types of Column Chromatography Based on Descriptions

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Which one of the following types of column chromatography correspond to the description 'only liquid chromatography method where molecules do NOT bind to the chromatography particles'.

 

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 '2D gels'.

 

Protein Net Charge at a Given pH

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Isoelectric Point Problem
Protein Name isoelectric point (pI) molecular weight
Leghemoglobin (Leg) 4.7 16.0

The protein in the table (above) is placed in a buffer solution with a pH of 6.0.
What is the correct net charge on the Leg protein at pH of 6.0

?
 

Protein Migration Direction from Isoelectric Point

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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
Succinate Ligase (SL) 6.6 20.9
Epidermal Growth Factor (EGF) 4.5 40.0

Both protein samples are placed into a gel with a constant pH of 5.5. The gel is then placed into an electric field.
In which direction will each protein in the table migrate at pH 5.5?

 

Calculating Molecular Weight from 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 a typical amount of time.
Standard ladder reference (kDa):

– 250
– 150
– 100
– 75
– 50
– 37
– 25
– 20
– 15
– 10

Gel results:

Lane 1
Lane 2

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)
Cytochrome c (Cyt) 13.0 3.45
Avidin (Av) 16.9 3.24
Succinate Ligase (SL) 20.9 3.07
Chymotrypsin (Chy) 25.0 2.92
Horseradish peroxidase (HP) 34.0 2.68
Epidermal Growth Factor (EGF) 40.0 2.55
Luciferase (Luc) 50.0 2.37
Unknown ? 3.15

Estimate the molecular weight of the unknown protein by comparing its gel migration distance with those of the standards.
 

Determining Isoelectric Point Using pKa

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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
CH3
H3N+
COOH
COOH
→
State 2
CH3
H3N+
COO−
COOH
→
State 3
CH3
H3N+
COO−
COO−
→
State 4
CH3
H2N
COO−
COO−
12
10
8
6
4
2
                      pKa2
                     
                       
                       
                      pKaR
                     
                       
                       
                      pKa1
                     
                       
                       
0 1 2 3  
OH− (equivalents)  

Given pKa values: pKa1 = 2.0, pKaR = 4.7, pKa2 = 9.1.
The isoelectric point (pI) is closest to which value?

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