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Questions and Answers
What is the primary principle behind SDS-PAGE in separating proteins?
What is the primary principle behind SDS-PAGE in separating proteins?
Which factor does NOT affect the migration of charged molecules during electrophoresis?
Which factor does NOT affect the migration of charged molecules during electrophoresis?
What is the role of Sodium Dodecyl Sulfate (SDS) in SDS-PAGE?
What is the role of Sodium Dodecyl Sulfate (SDS) in SDS-PAGE?
How does the sieving effect of the gel impact protein separation in SDS-PAGE?
How does the sieving effect of the gel impact protein separation in SDS-PAGE?
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In paper electrophoresis, how are the samples applied for separation?
In paper electrophoresis, how are the samples applied for separation?
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Which one of the following factors is crucial for the effectiveness of SDS-PAGE?
Which one of the following factors is crucial for the effectiveness of SDS-PAGE?
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What happens to proteins during SDS-PAGE due to the binding of SDS?
What happens to proteins during SDS-PAGE due to the binding of SDS?
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What type of molecules can be effectively separated using paper electrophoresis?
What type of molecules can be effectively separated using paper electrophoresis?
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What components are necessary for the polymerization of polyacrylamide gel?
What components are necessary for the polymerization of polyacrylamide gel?
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Which statement is true regarding the movement of DNA through agarose gel during electrophoresis?
Which statement is true regarding the movement of DNA through agarose gel during electrophoresis?
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What is the purpose of adding 6X sample loading buffer to DNA samples before gel electrophoresis?
What is the purpose of adding 6X sample loading buffer to DNA samples before gel electrophoresis?
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What role does the agarose gel play in DNA electrophoresis?
What role does the agarose gel play in DNA electrophoresis?
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In the context of polyacrylamide gel, what is bis-acrylamide used for?
In the context of polyacrylamide gel, what is bis-acrylamide used for?
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How does the size of the DNA affect its migration speed in an agarose gel during electrophoresis?
How does the size of the DNA affect its migration speed in an agarose gel during electrophoresis?
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What is a common component found in both staining and de-staining buffers for gels?
What is a common component found in both staining and de-staining buffers for gels?
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What factor is NOT directly affecting the speed of DNA migration in agarose gel electrophoresis?
What factor is NOT directly affecting the speed of DNA migration in agarose gel electrophoresis?
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What is the first step in the Southern blotting process?
What is the first step in the Southern blotting process?
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Which of the following methods is specifically for detecting RNA sequences?
Which of the following methods is specifically for detecting RNA sequences?
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During the Southern blotting process, what is used to visualize the DNA bands after detection?
During the Southern blotting process, what is used to visualize the DNA bands after detection?
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What is the purpose of the fixation step in Northern blotting?
What is the purpose of the fixation step in Northern blotting?
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What is the correct sequence of events for Northern blotting?
What is the correct sequence of events for Northern blotting?
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Which process involves the estimation of size and number of bands generated from DNA digestion?
Which process involves the estimation of size and number of bands generated from DNA digestion?
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What is the purpose of denaturing RNA in the Northern blotting process?
What is the purpose of denaturing RNA in the Northern blotting process?
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What is typically used as a solid support in Southern blotting?
What is typically used as a solid support in Southern blotting?
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What is the purpose of bromophenol blue in the loading buffer?
What is the purpose of bromophenol blue in the loading buffer?
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What direction does DNA migrate when an electrical current is applied?
What direction does DNA migrate when an electrical current is applied?
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How does bromophenol blue compare to a 300 bp DNA molecule during electrophoresis?
How does bromophenol blue compare to a 300 bp DNA molecule during electrophoresis?
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Which of the following is true regarding the staining process with ethidium bromide?
Which of the following is true regarding the staining process with ethidium bromide?
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What is the purpose of including a DNA ladder in the gel?
What is the purpose of including a DNA ladder in the gel?
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How long should the gel be allowed to stain in warm diluted ethidium bromide?
How long should the gel be allowed to stain in warm diluted ethidium bromide?
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What is a key safety precaution when handling ethidium bromide?
What is a key safety precaution when handling ethidium bromide?
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What should be done if excess stain remains on the gel after staining?
What should be done if excess stain remains on the gel after staining?
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Study Notes
Biomedical Techniques CLSB-222
- Course instructor: Dr. Ahmad Alamri, M.H.S, Ph.D
- Instructor email: [email protected]
Electrophoretic Techniques
- Electrophoresis is defined as the migration of charged molecules in an electric field
- It's a common technique in clinical laboratories for isolating and quantifying serum proteins, isoenzymes, hemoglobin, and lipoproteins
- Electrophoretic techniques are used in DNA sequencing, separation, and purification of biomolecules, and medical research
- Factors affecting electrophoresis include:
- Net electric charge on molecules (pH and ionic strength of buffer)
- Size and shape of molecules
- Electric field strength
- Nature of supporting media
- Temperature of operation
A) Paper Electrophoresis
- A method of electrophoresis performed on filter paper
- Useful for separating small charged molecules like amino acids and small proteins
- Samples are spotted on filter paper, and a high voltage is applied which causes molecules to migrate towards either the positive or negative pole based on charge.
- Different staining methods can be used to detect separated components based on chemical identity.
B) SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
- SDS-PAGE is a widespread method for protein separation in biochemistry, forensics, genetics, and molecular biology.
- It separates proteins based on their molecular weights.
- Principle: SDS is a detergent that denatures proteins, binds to hydrophobic regions, disrupts non-covalent bonds, and gives proteins a negative net charge, so proteins migrate based on their size.
Principle (Cont'd)
- Smaller proteins migrate faster than larger ones through the gel affected by electrical field strength
- The number of SDS molecules binding is proportional to protein size, thus, proteins in the electrical field migrate towards the anode (+) and are separated by molecular weight.
- During PAGE, the rate of migration of SDS-treated proteins is determined by molecular weight.
Polyacrylamide Gel
- Formed by the co-polymerization of acrylamide and N,N'-methylene-bis-acrylamide.
- To polymerize the gel, a system comprising ammonium persulfate (initiator) and tetramethylenediamine (TEMED) is added.
Staining the Gel
- Staining the gel with a buffer containing glacial acetic acid, methanol, and Coomassie brilliant blue 250-R, visualizing bands.
- Destaining the gel with a buffer containing glacial acetic acid and methanol to remove excess stain.
C) Agarose Gel Electrophoresis
- Used for separating DNA fragments based on size using agarose gel
- Separates DNA by rate of movement through gel affected by an electrical field.
- Useful in determining presence and size of PCR products.
- DNA is negatively charged.
- In electrophoresis, DNA migrates towards positive pole (anode).
- Agarose gel is used to slow down DNA movement and thus, separate DNA according to size.
DNA Ladder Standard
- Contains DNA fragments of known sizes.
- Used to determine the sizes of unknown DNA.
- Bromophenol blue on the gel migrates at approximately the same speed as 300 bp DNA.
Staining the Gel (Cont'd)
- Ethidium bromide binds to DNA, allowing visualization under UV light
- Ethidium bromide is a mutagen, thus, gloves must be worn.
Sample Preparation
- DNA samples are mixed with 6X sample loading buffer (bromophenol blue for color, glycerol for weight)
- This allows visualization of samples during loading and increases the sample density for better placement in gel wells.
- Carefully place pipette tip over well and gently expel the sample. Avoid puncturing the gel.
Running the Gel
- Place the cover on the electrophoresis chamber. Connect the electrical leads to the power supply ensuring DNA migrates to the positive/anode.
- Bubbles should form on the electrodes in the electrophoresis chamber when the power is turned on.
- Make sure the gel is running in the correct direction (DNA to positive pole). Bromophenol blue runs in the same direction as DNA.
D) Southern Blotting
- A technique for detecting specific DNA sequences in samples.
- Steps involve:
- Digestion of DNA with restriction enzymes
- Separating DNA fragments by agarose gel electrophoresis
- Denaturing DNA
- Transferring DNA to a solid support (nylon or nitrocellulose)
- Hybridizing immobilized DNA to a labeled probe (DNA or RNA)
- Detecting bands complementary to the probe (autoradiography)
- Estimating band size, and relative amount of target DNA
E) Northern Blotting
- A method to detect specific RNA sequences in samples
- Techniques include RNA extraction, electrophoresis, and hybridization using labeled probes
F) Western Blotting ("Immunoblotting")
- Separate and identify proteins on a gel
- Used to determine protein molecular weight and relative amounts.
- Method for detection of specific proteins by using antibodies
- Procedure involves gel electrophoresis, transfer to membrane, and detection
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Description
This quiz covers electrophoretic techniques utilized in biomedical labs, focusing on principles, applications, and factors influencing electrophoresis. Explore the nuances of paper electrophoresis and its significance in isolating biomolecules.
