Electrophoresis Flashcards
What is electrophoresis?
The migration of charged molecules in solution in response to an electric field
This process is often used in laboratories for separating biomolecules.
What is gel electrophoresis?
The migration of charged molecules through a gel under electric current
This technique is commonly used for DNA, RNA, and protein separation.
What is a major safety precaution when using electrophoresis equipment?
Use only electrophoresis equipment that is properly equipped with electrical interlocks to interrupt current flow when box is opened
This helps prevent electric shock hazards.
What is the purpose of electrophoresis?
To separate DNA, RNA or protein molecules
This separation allows for analysis and other applications in molecular biology.
What is a gel in the context of electrophoresis?
A Jello-like matrix with pores in it through which biological molecules will migrate
Gels are made of acrylamide or agarose.
What are agarose gels made from?
Seaweed, specifically a chain of sugar molecules
Agarose gels are commonly used for DNA separation.
What factors can influence the migration of molecules in gel electrophoresis?
Gel concentration, size and shape of the biomolecule, ionic strength of buffer
Each of these factors can affect how efficiently molecules move through the gel.
What is the difference between non-denaturing and denaturing polyacrylamide gels?
Non-denaturing (29:1) is used for double stranded DNA; Denaturing (19:1) is used for single stranded DNA
This distinction is critical for the type of analysis being performed.
What is Ohm’s law in the context of electrophoresis?
I(Current) = V(Voltage) / R(Resistance)
This law helps explain the relationship between current, voltage, and resistance during electrophoresis.
What is the role of ionic strength of the buffer in electrophoresis?
Conducts electric current by providing enough ions to sustain the electrical current for the entire run
Ionic strength must be maintained to ensure effective migration of molecules.
What is the purpose of loading dyes in gel electrophoresis?
Gives sample colour to see it run in the gel, allows for tracking of migration, contains ficoll or glycerol for density
Choosing the right loading dye is important to avoid interference with the sample.
What is a size ladder in gel electrophoresis?
A mixture of known fragments with known size used to estimate the size of molecules of interest
This is essential for determining the molecular weight of samples.
What are common visualization methods for DNA/RNA in gels?
- Ethidium Bromide (EtBr)
- SYBR Green
- SYBR Safe
- Gel Red
These methods help visualize nucleic acids in gels under UV light.
What is the most common method of protein detection in gels?
Staining with Coomassie dye
Coomassie G-250 or R-250 forms are typically used for this purpose.
What major protein groups are found in blood serum?
- Albumin
- α1 Globulin
- α2 Globulin
- β Globulin
- γ Globulin
These proteins have various functions, including maintaining blood volume and transporting substances.
What is the pH used in serum protein electrophoresis?
8.6
At this pH, proteins carry a net negative charge and migrate towards the positive end.
Fill in the blank: The _______ is the most common method of in-gel protein detection.
Coomassie dye
This method is widely used due to its effectiveness and ease of use.
True or False: Agarose gels can separate DNA fragments from 100bp to 30kb.
True
This range makes agarose gels versatile for various DNA applications.
What are the disadvantages of using constant current during gel electrophoresis?
- Increasing heat over time
- Harder to run multiple gels off of the same power pack
These factors can complicate the electrophoresis process.
How does the concentration of agarose affect gel density?
Higher concentration results in smaller pores, slowing the migration of larger fragments
This is critical for optimizing separation based on fragment size.
What happens to the ionic strength of the buffer during electrophoresis?
It decreases, increasing resistance
This can affect the efficiency of the electrophoresis run.
What is serum protein electrophoresis performed with?
A standard barbital buffer with a standard ionic strength of 0.05 and a pH of 8.6
At this pH, proteins have a net negative charge and migrate to the positive end.
How much serum is applied to the gel during serum protein electrophoresis?
3-5uL
What is the duration for applying constant voltage during serum protein electrophoresis?
40-60 min
What is used to fix the bands after electrophoresis?
A fixative (MetOH and Acetic acid)
What stain is used for visualization in serum protein electrophoresis?
Coomassie blue
How many bands are typically present in a normal serum protein electrophoresis?
5 bands
What phenomenon affects the motion of macromolecules during protein gel electrophoresis?
Electroendosmosis
True or False: In serum protein electrophoresis, the gamma globulin band appears after the point of sample application.
False
What is the purpose of immunoelectrophoresis?
To separate antigens and determine the type of antigen after electrophoresis
In immunofixation electrophoresis, how many lanes are typically used?
6 lanes
What is SDS-PAGE?
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis
What does SDS do to proteins during SDS-PAGE?
Denatures proteins and binds to them, conferring a net negative charge
What is the purpose of Western Blots?
To detect specific proteins after separation by SDS-PAGE
What is Blue Native PAGE used for?
To analyze proteins in their native form
What is the main component used to confer a negative charge in Blue Native PAGE?
Coomassie
What is the primary method of detecting samples in capillary electrophoresis?
Optical detection (e.g., fluorescence)
What two properties are proteins separated by in 2D SDS-PAGE?
- Isoelectric point
- Mass
Fill in the blank: In capillary electrophoresis, _______ is the motion of liquid when a voltage is applied.
Electroosmosis
What affects the speed of migration through capillary electrophoresis?
The size of the molecule
What are the advantages of capillary electrophoresis?
- Automated
- Higher resolution
- Less toxic
- Short analytical time
- Smaller sample volumes
What is the purpose of using a size ladder in electrophoresis?
To determine the exact size of the molecule of interest
List the techniques that rely on gel electrophoresis.
- Northern Blot
- Southern Blot
- Eastern Blot
- Western Blot
- Sanger sequencing
- RFLP analysis
