MCAT protein purification and electrophoresis review
This flashcard set covers essential concepts of protein purification techniques and electrophoresis methods relevant for the MCAT, focusing on high-yield facts and relationships critical for success.
Quiz(40 questions)
1. What is the main purpose of electrophoresis?
Terms in this Study Set(40)
Protein Purification Techniques(20)
What is the purpose of protein purification?
To isolate a specific protein from a complex mixture for analysis.
Define chromatography in protein purification.
A technique that separates proteins based on size, charge, or affinity.
True or False: Centrifugation is used to separate proteins by charge.
False - Centrifugation separates based on density and size.
Fill in the blank: Affinity chromatography uses _______ to bind specific proteins.
ligands or antibodies
Compare ion exchange and size exclusion chromatography.
Ion exchange: separates by charge. Size exclusion: separates by size.
What is the role of a buffer in chromatography?
Maintains pH and ionic strength for optimal separation.
How does centrifugation work?
Spins samples to create a centrifugal force, separating components by density.
What does SDS-PAGE stand for?
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
True or False: High-performance liquid chromatography (HPLC) is efficient for protein purification.
True - HPLC offers high resolution and speed.
What is a common method for concentrating proteins?
Ultrafiltration, using membranes to filter out smaller molecules.
List two types of affinity chromatography.
- Histidine-tagged proteins - Antibody affinity purification
Cause → Effect: Increased salt concentration in ion exchange chromatography.
Improved elution of bound proteins.
Describe the principle of size exclusion chromatography.
Larger molecules pass through faster, while smaller ones are delayed.
What is the role of gel electrophoresis?
To separate proteins based on their size and charge.
Fill in the blank: Protein purification often requires _______ to assess purity.
SDS-PAGE analysis
Define affinity tags in protein purification.
Short peptide sequences that facilitate specific binding to capture proteins.
How does molecular weight affect protein migration in gel electrophoresis?
Larger proteins migrate slower than smaller proteins.
What is the main advantage of using a FPLC system?
It allows for continuous flow and automated fraction collection.
What is the role of detergents in protein purification?
They solubilize membranes and denature proteins for better separation.
True or False: More purification steps always lead to higher yield.
False - More steps can decrease overall yield.
Electrophoresis Fundamentals(20)
What is electrophoresis?
A technique to separate charged molecules, primarily proteins, based on their size and charge.
True or False: Electrophoresis can only separate DNA.
False. It can separate proteins, nucleic acids, and other charged biomolecules.
How does the charge of a protein affect its movement?
Positively charged proteins move towards the cathode; negatively charged proteins move towards the anode.
Fill in the blank: In gel electrophoresis, molecules migrate through a __________.
gel matrix, typically agarose or polyacrylamide.
Comparison: SDS-PAGE vs. native PAGE.
- SDS-PAGE denatures proteins and coats them with negative charge. - Native PAGE retains protein structure and charge.
What does the term 'molecular weight standard' mean?
A set of known proteins used as a reference to estimate the size of other proteins.
Cause → Effect: Increasing voltage during electrophoresis.
Increases speed of migration, potentially leading to heat generation and band distortion.
What role does the buffer play in electrophoresis?
Maintains pH and ionic strength, ensuring consistent charge carriers for molecule migration.
True or False: Proteins with the same charge will not separate in electrophoresis.
True. Separation is based on size; identical charges will not lead to separation.
Name one application of electrophoresis in research.
Protein characterization, quantification, or purity assessment.
How does pH affect protein charge?
Altering pH can change the ionization state of amino acids, affecting overall charge.
What is the purpose of a loading dye?
To track sample migration and visualize bands during electrophoresis.
Fill in the blank: The direction of migration in electrophoresis is dependent on the __________ of the molecules.
net charge and size.
What is the significance of isoelectric focusing?
Separates proteins based on their isoelectric point (pI), where they have no net charge.
Comparison: Agarose gel vs. polyacrylamide gel.
- Agarose: Best for DNA/RNA, larger pores. - Polyacrylamide: Best for proteins, smaller pores.
How can you visualize proteins after electrophoresis?
Commonly by staining methods such as Coomassie Blue or silver staining.
Why use a gradient gel in electrophoresis?
To separate proteins of varying sizes more effectively by creating a range of pore sizes.
What does 'smearing' indicate on a gel?
It can suggest protein degradation, multiple isoforms, or incomplete separation.
True or False: Electrophoresis can be performed in a non-aqueous medium.
True. Non-aqueous electrophoresis is used for certain special applications.
What does the term 'electrophoretic mobility' refer to?
The velocity of a charged particle in an electric field, influenced by charge and size.
Questions in this Study Set(40)
1. What is the main purpose of electrophoresis?
2. What is the primary goal of protein purification?
3. Which type of gel is best for separating smaller proteins?
4. Which technique separates proteins based on their electric charge?
5. True or False: In isoelectric focusing, proteins are separated based on their size.
6. True or False: Gel filtration chromatography is another name for size exclusion chromatography.
7. Which factor does NOT affect the migration speed of proteins in electrophoresis?
8. What is the function of ligands in affinity chromatography?
9. What is the role of a buffer in gel electrophoresis?
10. In which scenario would you use size exclusion chromatography?
11. Fill in the blank: In SDS-PAGE, proteins are coated with __________.
12. Which of the following is NOT a method for protein purification?
13. What does a molecular weight standard do in electrophoresis?
14. What does the term 'elution' refer to in chromatography?
15. True or False: Proteins with the same molecular weight will always have the same migration distance in electrophoresis.
16. What is the purpose of a buffer in chromatography?
17. What happens to proteins if the voltage is increased during electrophoresis?
18. How does molecular weight affect protein separation in SDS-PAGE?
19. Which is NOT a common staining method used after electrophoresis?
20. What is one major advantage of using High-Performance Liquid Chromatography (HPLC)?
21. How does increasing gel concentration affect pore size?
22. Fill in the blank: The technique that separates proteins by their size is called _______.
23. What is the significance of using a loading dye?
24. What is the role of detergents in protein purification?
25. Which type of electrophoresis is best for separating nucleic acids?
26. True or False: Affinity tags are only used for purification of native proteins.
27. What does 'smearing' on a gel typically indicate?
28. Which method is commonly used to concentrate proteins?
29. True or False: Electrophoresis can be performed in a vacuum.
30. What is the effect of increased salt concentration in ion exchange chromatography?
31. Which factor is crucial for maintaining the ionic strength during electrophoresis?
32. What does SDS-PAGE stand for?
33. What does electrophoretic mobility depend on?
34. Which of the following statements is true regarding protein purification?
35. Which is NOT a characteristic of native PAGE?
36. Which of the following techniques separates proteins based on their size without regard to charge?
37. Which of the following statements correctly describes the effect of pH on protein charge during electrophoresis?
38. What is one key advantage of using affinity chromatography in protein purification?
39. In a gel electrophoresis experiment, which gel type would be least suitable for resolving very large proteins?
40. Which of the following describes the main function of SDS in SDS-PAGE?
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