Electrophoresis and Applications — MCQs
What is Northern blot used to detect?
Which of the following separates proteins solely on the basis of their molecular size?
Which among the following is identified by Western blotting
Which of the following is the most important diagnostic investigation for multiple myeloma?
Which technique is used for protein separation based on molecular size?
Diagnosis of beta thalassemia is established by what?
Which technique is used for the separation of proteins based on their mass?
The shown pattern in electrophoresis is due to:
The technique shown in the image is:
Separation of proteins based on size is done by
Electrophoresis and Applications Indian Medical PG Practice Questions and MCQs
Question 1: What is Northern blot used to detect?
- A. Protein
- B. Immunoglobulin
- C. RNA (Correct Answer)
- D. DNA
Explanation: ***RNA*** - **Northern blot** is a laboratory technique used to detect specific **RNA** molecules among a mixture of RNA. - It involves separating RNA fragments by **gel electrophoresis**, transferring them to a membrane, and then probing with a labeled complementary sequence. *Protein* - **Proteins** are typically detected using a **Western blot**, which involves similar separation and transfer techniques but uses **antibodies** as probes. - While RNA codes for proteins, Northern blot *directly* detects RNA transcripts, not the resulting protein products. *Immunoglobulin* - **Immunoglobulins** (antibodies) are a type of protein, and their detection usually falls under **Western blot** or specific immunological assays like **ELISA**. - Northern blot is specifically designed for nucleic acid analysis, not protein detection. *DNA* - **DNA** is detected using a **Southern blot** technique, which also involves electrophoresis, transfer to a membrane, and hybridization with a complementary probe. - The name "Northern blot" was coined as a play on "Southern blot" because it uses similar methodology but for RNA instead of DNA.
Question 2: Which of the following separates proteins solely on the basis of their molecular size?
- A. Isoelectric focusing
- B. Chromatography on a diethylaminoethyl (DEAE) cellulose column
- C. Gel filtration chromatography (Correct Answer)
- D. Chromatography on a carboxymethyl (CM) cellulose column
Explanation: ***Gel filtration chromatography*** - Also known as **size-exclusion chromatography**, this method separates proteins based on their **hydrodynamic volume** (molecular size and shape). - Larger proteins pass through the column more quickly because they are excluded from the pores of the stationary phase, while smaller proteins enter the pores and have a longer, more tortuous path. *Isoelectric focusing* - This technique separates proteins based on their **isoelectric point (pI)**, which is the pH at which the protein has no net electrical charge. - Proteins migrate through a pH gradient until they reach the point where their net charge is zero. *Chromatography on a diethylaminoethyl (DEAE) cellulose column* - **DEAE cellulose** is an **anion-exchange resin**, meaning it binds **negatively charged** proteins. - Separation is based on the **net charge** of the protein at a given pH. *Chromatography on a carboxymethyl (CM) cellulose column* - **CM cellulose** is a **cation-exchange resin**, meaning it binds **positively charged** proteins. - Separation is based on the **net charge** of the protein at a given pH.
Question 3: Which among the following is identified by Western blotting
- A. t-RNA
- B. RNA
- C. DNA
- D. Proteins (Correct Answer)
Explanation: ***Proteins*** - **Western blotting** is a widely used analytical technique in molecular biology and immunogenetics to detect specific **proteins** in a sample. - The technique involves separating proteins by gel electrophoresis, transferring them to a membrane, and then detecting the target protein using specific antibodies. *t-RNA* - **t-RNA** (transfer ribonucleic acid) is involved in protein synthesis but is not typically detected using Western blotting. - While other blotting techniques exist for RNA, Western blotting is specific for protein analysis, not for detecting different types of RNA. *RNA* - General **RNA** detection is usually performed using techniques like **Northern blotting** or RT-PCR, not Western blotting. - Western blotting relies on antibody-antigen interactions specific to protein structures. *DNA* - **DNA** is detected using techniques such as **Southern blotting** or PCR, not Western blotting. - Western blotting is designed to identify proteins based on their molecular weight and antigenicity.
Question 4: Which of the following is the most important diagnostic investigation for multiple myeloma?
- A. Lytic bone lesions
- B. Bence jones proteins
- C. Alkaline Phosphatase
- D. Serum electrophoresis (Correct Answer)
Explanation: ### Serum electrophoresis - **Serum protein electrophoresis** (SPEP) is crucial for detecting and quantifying the **monoclonal paraprotein (M-protein)** in the blood, which is characteristic of multiple myeloma [1]. - The presence of a **gamma globulin spike** or **M-spike** on SPEP is a hallmark of the disease [1]. *Lytic bone lesions* - While **lytic bone lesions** are a common and important feature of multiple myeloma, they are a consequence of the disease process rather than the primary diagnostic investigation itself [1]. - Imaging studies like X-rays or MRI are used to detect these lesions, but their presence alone is not sufficient for diagnosis without identification of the M-protein [1]. *Bence jones proteins* - **Bence Jones proteins** are **monoclonal light chains** found in the urine, indicating their presence in the blood. - While important for diagnosis and prognosis, SPEP is generally considered more central as it directly identifies the **monoclonal gammopathy** (M-protein) derived from plasma cells in the serum [1]. *Alkaline Phosphatase* - **Alkaline phosphatase** levels are typically **normal** in multiple myeloma, even in the presence of extensive bone disease. - This is because the lytic lesions in multiple myeloma result from osteoclast activation rather than osteoblastic activity, which would otherwise elevate alkaline phosphatase.
Question 5: Which technique is used for protein separation based on molecular size?
- A. Affinity chromatography
- B. Gel filtration chromatography (Correct Answer)
- C. HPLC
- D. Salting out
Explanation: ***Gel filtration chromatography*** - Also known as **size-exclusion chromatography**, this method separates proteins by passing them through a porous matrix. **Larger proteins** elute first as they cannot enter the pores, while smaller proteins get trapped and elute later. - This technique effectively separates proteins based solely on their **hydrodynamic radius**, which is closely related to their molecular size. *Affinity chromatography* - This method separates proteins based on their **specific binding affinity** to a ligand immobilized on a stationary phase, not molecular size. - It is used for purifying proteins that bind to a specific molecule, such as an antibody or substrate. *HPLC* - **High-performance liquid chromatography** is a general technique that can use various separation mechanisms (e.g., reverse-phase, ion-exchange, size-exclusion) under high pressure. - While it *can* be used for size-exclusion, HPLC itself describes the *method* of chromatographic performance rather than a specific separation principle based on molecular size alone. *Salting out* - This technique separates proteins based on their **solubility** in high salt concentrations. - As salt concentration increases, the proteins lose their hydration shells and precipitate out of solution, with different proteins precipitating at different salt concentrations.
Question 6: Diagnosis of beta thalassemia is established by what?
- A. Hb electrophoresis (Correct Answer)
- B. NESTROFT screening test
- C. Hemoglobin A1c test
- D. Presence of target cells in blood smear
Explanation: Hb electrophoresis - Hemoglobin electrophoresis directly measures the relative proportions of different hemoglobin types (HbA, HbA2, HbF), which is crucial for identifying the characteristic reduction in HbA and elevation of HbA2 and HbF in beta thalassemia. [1] - This method provides a definitive diagnostic profile by separating hemoglobin based on their electrical charge and size, allowing for quantification of abnormal hemoglobin variants. [1] *NESTROFT screening test* - The NESTROFT (Naked Eye Single Tube Red cell Osmotic Fragility Test) is a screening tool used to identify individuals with thalassemia traits and is not a definitive diagnostic test. - While useful for mass screening due to its simplicity and cost-effectiveness, it requires confirmation with more specific tests like hemoglobin electrophoresis. [1] *Hemoglobin A1c test* - The Hemoglobin A1c (HbA1c) test is primarily used to monitor long-term blood glucose control in individuals with diabetes. [2] - It measures the percentage of hemoglobin glycated over a period of 2-3 months and has no direct diagnostic utility for thalassemia. [2] *Presence of target cells in blood smear* - The presence of target cells in a blood smear is a non-specific finding that can be observed in various conditions, including iron deficiency anemia, liver disease, and other hemoglobinopathies, in addition to thalassemia. - While suggestive of a thalassemic disorder, it is not a conclusive diagnostic criterion and requires further investigation with specific diagnostic tests.
Question 7: Which technique is used for the separation of proteins based on their mass?
- A. Electrophoresis
- B. Salting out
- C. SDS-PAGE (Correct Answer)
- D. Ion exchange chromatography
Explanation: ***Correct Option: SDS-PAGE*** - **SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)** separates **denatured proteins** almost exclusively by their **molecular mass**. - **SDS** binds to proteins, imparting a uniform negative charge-to-mass ratio, ensuring that separation is primarily based on their size as they migrate through a **polyacrylamide gel**. - This is the gold standard technique for analyzing proteins by molecular weight. *Incorrect Option: Electrophoresis* - This is a general technique that uses an **electric field** to separate molecules based on their **charge** and **size**. - While it can separate proteins, it doesn't exclusively rely on **mass** without additional modifications (like SDS). - Native electrophoresis separates by charge-to-mass ratio, not mass alone. *Incorrect Option: Salting out* - This technique separates proteins based on their **solubility** in high salt concentrations. - Proteins "salt out" or precipitate at different salt concentrations, which is not directly related to their **mass**. - Based on protein surface properties and hydrophobicity. *Incorrect Option: Ion exchange chromatography* - This method separates proteins based on their **net charge** at a particular pH. - Proteins bind to a charged resin and are eluted by changing the **ionic strength** or **pH** of the buffer. - Two types: cation exchange (negative resin) and anion exchange (positive resin).
Question 8: The shown pattern in electrophoresis is due to:
- A. Charges (Correct Answer)
- B. Molecular weight
- C. Bound oxygen
- D. Hydrophobicity
Explanation: ***Charges*** - Electrophoresis separates molecules, such as **hemoglobin variants**, based on their **electrical charge** when placed in an electric field. - Different amino acid substitutions in hemoglobin lead to changes in net charge, causing them to migrate at different rates in the gel, as seen by the distinct bands for Hb A, Hb S/D, and Hb A2. *Molecular weight* - While molecular weight can influence migration in some electrophoretic techniques (e.g., SDS-PAGE), standard hemoglobin electrophoresis primarily separates based on **charge differences**, not molecular size. - All common hemoglobin variants have a very similar **molecular weight** (approximately 64,500 Da), so this factor would not effectively separate them into distinct bands. *Bound oxygen* - The amount of **bound oxygen** to hemoglobin does not significantly alter its overall electrical charge or molecular weight to cause distinct bands in electrophoresis. - Oxygen binding is a dynamic process and does not account for the **structural differences** in hemoglobin variants that lead to their electrophoretic separation. *Hydrophobicity* - Hydrophobicity is a characteristic of molecules, but it is not the primary principle by which standard **gel electrophoresis** separates hemoglobin variants. - Techniques like **hydrophobic interaction chromatography** would exploit hydrophobicity, not the electrophoretic gel shown.
Question 9: The technique shown in the image is:
- A. High performance liquid chromatography (Correct Answer)
- B. Haemoglobin electrophoresis
- C. Gel electrophoresis
- D. Tandem mass spectrometry
Explanation: ***High performance liquid chromatography*** - The image displays a **chromatogram** with distinct peaks labeled HbA1c, HbF, HbA0, and HbA2, separated based on their chemical properties. - This separation and detection method is characteristic of **High Performance Liquid Chromatography (HPLC)**, a technique used for quantifying different hemoglobin fractions. *Tandem mass spectrometry* - **Tandem mass spectrometry (MS/MS)** identifies compounds based on their mass-to-charge ratio and fragmentation patterns, which would look like mass spectra, not peaks on a time-based chromatogram. - While MS/MS is highly sensitive and specific, it doesn't produce the type of **elution profile** seen in the image. *Haemoglobin electrophoresis* - **Hemoglobin electrophoresis** separates hemoglobins based on their electrical charge, resulting in bands on a gel or a densitometric scan, not the **distinct chromatogram peaks** shown here. - While used for hemoglobin analysis, the visual representation is typically different, often displaying bands that reflect migration distance. *Gel electrophoresis* - **Gel electrophoresis** separates molecules, such as proteins or nucleic acids, by size and charge through a gel matrix, producing distinct **bands** that can be visualized. - This method would not produce the continuous **elution peaks over time** as observed in the provided graph, which indicates a liquid chromatography technique.
Question 10: Separation of proteins based on size is done by
- A. Affinity chromatography
- B. High performance liquid chromatography
- C. SDS-Polyacrylamide gel electrophoresis (Correct Answer)
- D. Ion exchange chromatography
Explanation: ***SDS-Polyacrylamide gel electrophoresis*** - **SDS-PAGE** separates proteins primarily based on their **molecular weight** (size). - Proteins are denatured and coated with negatively charged **SDS**, causing them to migrate through a polyacrylamide gel based on size. *Affinity chromatography* - This technique separates proteins based on their **specific binding affinity** to a ligand. - It does not directly separate based on size, but rather on **molecular recognition**. *High performance liquid chromatography* - **HPLC** is a chromatographic technique that separates molecules in a complex mixture, but the primary basis of separation depends on the column type. - While some HPLC methods (**size-exclusion HPLC**) can separate by size, it is a broader technique and not the most specific answer for protein size separation in general context. *Ion exchange chromatography* - This method separates proteins based on their **net charge** at a particular pH. - Proteins bind to a charged resin and are eluted by increasing salt concentration or changing pH, not based on size.
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