Biochemical Techniques Practice Questions

Q: Which of the following separates proteins solely on the basis of their molecular size?

Gel filtration chromatography. ***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.

Q: Northern blot technique is used in the detection of

RNA. ***RNA*** - The **Northern blot** is a laboratory technique used to study **gene expression** by detecting specific **RNA molecules** (mRNA, rRNA, or tRNA) in a sample. - It involves separating RNA fragments by size via gel electrophoresis, transferring them to a membrane, and then probing with a labeled nucleic acid sequence complementary to the target RNA. *Protein* - The detection of **proteins** is typically performed using a **Western blot** technique, which identifies specific proteins through antibody binding. - While RNA ultimately codes for proteins, Northern blotting directly analyzes the **RNA transcript** itself, not the protein product. *DNA* - **Southern blot** is the technique specifically designed for the detection of **DNA sequences**, used to analyze genomic DNA or specific DNA fragments. - Northern blot differentiates itself from Southern blot by its application in analyzing **gene expression at the RNA level**. *plasmid* - A **plasmid** is a small, circular, extrachromosomal **DNA molecule** found in bacteria and some other organisms. - Detection of plasmids, being DNA, would primarily involve techniques like **Southern blot** or **PCR**, not Northern blot which targets RNA.

Q: Protein Electrophoresis (PE) is a type of:-

Electrophoretic technique. ***Electrophoretic technique*** - **Protein electrophoresis (PE)** is fundamentally an **electrophoretic separation technique** that uses an **electric field** to separate charged protein molecules based on their **size, charge, and shape** - The core principle involves **migration of charged particles** through a medium (gel or capillary) under the influence of an electric field - Common types include **SDS-PAGE**, **native PAGE**, **isoelectric focusing**, and **capillary electrophoresis** - This is the standard classification in all major biochemistry textbooks (Harper's, Lehninger) *Chromatographic technique* - **Chromatography** separates molecules based on differential partitioning between a **stationary phase** and a **mobile phase** (not an electric field) - Examples include **HPLC**, **gas chromatography**, and **column chromatography** - Different separation principle from electrophoresis *Immunological technique* - **Immunological techniques** primarily use **antigen-antibody interactions** for detection, such as **ELISA**, **immunoprecipitation**, or **Western blot** - While **immunofixation electrophoresis** combines both principles, standard PE does not rely on immunological reactions for separation *Spectroscopic technique* - **Spectroscopy** involves studying the interaction between matter and **electromagnetic radiation** (e.g., **UV-Vis spectrophotometry**, **NMR**, **mass spectrometry**) - While electrophoresis results may be **detected** using spectroscopic methods (like densitometry after staining), the separation technique itself is electrophoretic, not spectroscopic - Detection method ≠ separation technique

Q: Northern Blot is used to detect

RNA. ***RNA*** - Northern blot analysis is a laboratory technique used to detect specific **RNA sequences** in a sample. - This method involves **electrophoresis** to separate RNA fragments by size, followed by transfer to a membrane and hybridization with a labeled RNA or DNA probe. *DNA* - **Southern blotting** is the technique specifically designed for detecting **DNA sequences**. - It involves digesting DNA with restriction enzymes, separating fragments by electrophoresis, and hybridizing with a labeled DNA probe. *Proteins* - **Western blotting** is the technique used for detecting and analyzing **proteins**. - Proteins are separated by size using SDS-PAGE, transferred to a membrane, and then probed with specific antibodies. *Enzymes* - While enzymes are proteins, directly detecting **enzyme activity** or specific enzymes often involves **activity assays** or **immunological methods** like ELISA, rather than Northern blotting. - Northern blotting identifies mRNA transcripts, which are templates for protein (including enzyme) synthesis, but not the enzymes themselves.

Q: Which of the following is NOT true about hybridoma?

In-situ hybridization. ***In-situ hybridization*** - Hybridoma technology is used to produce **monoclonal antibodies**, not for **in-situ hybridization**, which is a molecular biology technique for detecting specific nucleic acid sequences within cells or tissues. - While antibodies produced by hybridomas can be used in immunohistochemistry (a related technique), the hybridoma process itself does not involve **in-situ hybridization**. *DNA sequencing* - **DNA sequencing** is a technique to determine the order of nucleotide bases in a DNA molecule. - Hybridoma technology is focused on producing antibodies from fused cells, not on directly sequencing DNA, though antibodies could be used in research to study DNA-binding proteins. *Formation of continuous cell line* - One of the primary advantages of hybridoma technology is the creation of a **continuous cell line** by fusing antibody-producing B cells with myeloma cells. - This immortalized cell line allows for the consistent and long-term production of specific antibodies. *Formation of monoclonal antibodies* - The main purpose of the hybridoma technique is the production of **monoclonal antibodies**, which are highly specific antibodies that recognize a single epitope. - These antibodies have broad applications in diagnostics, therapeutics, and research.

Q: The fastest moving amino acid in methanol-chloroform electrophoresis is:

Glycine. ***Glycine*** - In **methanol-chloroform electrophoresis** (a **non-polar solvent system**), amino acids exist primarily in their **non-ionized forms**, unlike aqueous electrophoresis. - Migration depends on **molecular size and polarity** rather than ionic charge. - **Glycine** is the **smallest amino acid** (molecular weight 75 Da) with minimal side chain interactions, allowing it to move fastest through the non-polar medium. - Its simple structure (H as side chain) provides the least resistance to movement in organic solvents. *Aspartic Acid* - **Aspartic acid** is an acidic amino acid that would be in its **non-ionized form** in the non-polar methanol-chloroform system. - Its **larger size** and **polar carboxyl groups** create more interactions with the solvent, slowing its migration. - The concept of "net negative charge" does not apply in non-polar solvents where ionization is minimal. *Valine* - **Valine** is a **branched-chain, hydrophobic amino acid** with a larger side chain than glycine. - While it is non-polar and compatible with the solvent, its **larger molecular size** (117 Da) results in slower migration compared to glycine. *Leucine* - **Leucine** is also a **branched-chain, hydrophobic amino acid** with an even larger side chain than valine (131 Da). - Its **larger molecular size** and bulkier structure create more resistance to movement through the electrophoretic medium, making it the slowest among the options.

Q: Column chromatography using phenyl Sepharose beads separates proteins based on the

Hydrophobicity. ***Hydrophobicity*** - Phenyl Sepharose beads are designed with **hydrophobic phenyl groups** covalently linked to the Sepharose matrix. - Proteins with higher inherent hydrophobicity interact more strongly with these phenyl groups, leading to **retention on the column** and separation based on their **hydrophobic tendencies**. *Charge* - Separation based on charge typically employs **ion-exchange chromatography**, which uses charged resins to bind proteins based on their overall net charge. - Phenyl Sepharose chromatography does **not primarily separate** proteins based on their electrostatic properties. *Stokes' radius* - Separation based on Stokes' radius (or molecular size) is achieved using **size-exclusion chromatography** (or gel filtration chromatography), where porous beads fractionate proteins based on their ability to enter the pores. - Phenyl Sepharose chromatography does **not primarily rely on size exclusion** for protein separation. *Affinity to ligand* - Separation based on affinity to a specific ligand is characteristic of **affinity chromatography**, where a ligand is immobilized on the matrix to specifically bind a target protein. - While phenyl groups can be considered ligands, their interaction is general hydrophobic, not specific to a particular protein-ligand binding site.

Q: In estimation of bilirubin, the solution is examined at

450 nm. ***450 nm*** - **Bilirubin** absorbs light most strongly at a wavelength of approximately **450 nm**, which is in the **blue-violet region** of the visible spectrum. - This wavelength is used in the **Jendrassik-Gróf method** and other **spectrophotometric methods** for accurate estimation of bilirubin concentration. *540 nm* - This wavelength is typically used in assays for metabolites such as **glucose** and **uric acid**, often after a colorimetric reaction. - It is not the optimal absorption maximum for bilirubin and would lead to less accurate readings. *480 nm* - While close to the bilirubin absorption peak, **480 nm** is not the maximal absorption wavelength and would result in reduced sensitivity and accuracy. - Using a wavelength slightly off the maximum peak means that not all of the light absorbed will be due to bilirubin, introducing error. *580 nm* - This wavelength is too far from the peak absorption of bilirubin, which is in the blue-violet region. - Using **580 nm** would result in very little light absorption by **bilirubin**, making it unsuitable for quantitative analysis.

Q: A chemical is tested for carcinogenicity by examining its mutagenic effects on bacterial cells in culture. Which of the following tests is used to make this determination?

Ames test. ***Ames test*** - The **Ames test** is a widely used biological assay to assess the **mutagenic potential** of chemical compounds. - It uses specific strains of bacteria (e.g., *Salmonella typhimurium* or *Escherichia coli*) that have been genetically modified to require a particular nutrient (e.g., histidine) for growth and examines the frequency of **reverse mutations** that allow them to grow without that nutrient, indicating mutagenicity. *Watson-Schwartz test* - The **Watson-Schwartz test** is a biochemical test used to detect the presence of **porphobilinogen** in urine, primarily for diagnosing acute intermittent porphyria. - It is not related to assessing mutagenicity or carcinogenicity. *Widal test* - The **Widal test** is a serological test used for the diagnosis of **typhoid fever** by detecting antibodies against *Salmonella typhi* O and H antigens in a patient's serum. - It is an immunological test and does not assess mutagenic effects. *Nitroblue tetrazolium test* - The **Nitroblue tetrazolium (NBT) test** is used to assess the phagocytic function of **neutrophils**, primarily to diagnose **chronic granulomatous disease (CGD)**. - It measures the ability of neutrophils to produce **superoxide radicals** and is not related to carcinogenicity or mutagenicity.

Q: Ammonium oxalate reagent is commonly used in analytical chemistry for the detection of:

Calcium. ***Calcium*** - Ammonium oxalate is the **standard reagent** used for the qualitative and quantitative detection of **calcium ions** (Ca²⁺) in analytical chemistry. - It forms a **white crystalline precipitate** of calcium oxalate (CaC₂O₄·H₂O) which is insoluble in water and acetic acid. - This is a **classical precipitation reaction** widely used in clinical laboratories for calcium estimation and in urine analysis for detecting calcium oxalate crystals. - The reaction is: Ca²⁺ + (NH₄)₂C₂O₄ → CaC₂O₄↓ + 2NH₄⁺ *Ammonium molybdate* - This compound is itself a *reagent* used for detecting **phosphate ions**, not a substance detected by ammonium oxalate. - It forms yellow ammonium phosphomolybdate complex in the presence of phosphates under acidic conditions. *Sodium phosphomolybdate* - This is a *product formed* in phosphate detection tests using ammonium molybdate. - It is not detected by ammonium oxalate, which is specific for calcium ion detection. *Magnesium* - Magnesium ions are detected using a *different reagent system* - typically disodium hydrogen phosphate (Na₂HPO₄) in the presence of ammonium hydroxide. - This forms magnesium ammonium phosphate (struvite) precipitate, **not** a reaction with ammonium oxalate. - Ammonium oxalate does not form a characteristic precipitate with magnesium ions.

Question 1

Which of the following separates proteins solely on the basis of their molecular size?

Accepted Answer

Gel filtration chromatography

Answer

Isoelectric focusing

Answer

Chromatography on a diethylaminoethyl (DEAE) cellulose column

Answer

Chromatography on a carboxymethyl (CM) cellulose column

Question 2

Northern blot technique is used in the detection of

Accepted Answer

RNA

Answer

Protein

Answer

DNA

Answer

plasmid

Question 3

Protein Electrophoresis (PE) is a type of:-

Accepted Answer

Electrophoretic technique

Answer

Chromatographic technique

Answer

Immunological technique

Answer

Spectroscopic technique

Question 4

Northern Blot is used to detect

Accepted Answer

RNA

Answer

DNA

Answer

Proteins

Answer

Enzymes

Question 5

Which of the following is NOT true about hybridoma?

Accepted Answer

In-situ hybridization

Answer

DNA sequencing

Answer

Formation of continuous cell line

Answer

Formation of monoclonal antibodies

Question 6

The fastest moving amino acid in methanol-chloroform electrophoresis is:

Accepted Answer

Glycine

Answer

Valine

Answer

Leucine

Answer

Aspartic Acid

Question 7

Column chromatography using phenyl Sepharose beads separates proteins based on the

Accepted Answer

Hydrophobicity

Answer

Charge

Answer

Stokes' radius

Answer

Affinity to ligand

Question 8

In estimation of bilirubin, the solution is examined at

Accepted Answer

450 nm

Answer

540 nm

Answer

480 nm

Answer

580 nm

Question 9

A chemical is tested for carcinogenicity by examining its mutagenic effects on bacterial cells in culture. Which of the following tests is used to make this determination?

Accepted Answer

Ames test

Answer

Watson-Schwartz test

Answer

Widal test

Answer

Nitroblue tetrazolium test

Question 10

Ammonium oxalate reagent is commonly used in analytical chemistry for the detection of:

Accepted Answer

Calcium

Answer

Ammonium molybdate

Answer

Sodium phosphomolybdate

Answer

Magnesium

Biochemical Techniques — MCQs

Biochemical Techniques — MCQs

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