Biochemical Techniques Practice Questions

Q: The following method of chromatography is called:

Sephadex chromatography. ***Sephadex chromatography*** - The image depicts a column filled with beads (pink circles) that act as a **porous stationary phase**. Smaller molecules (black dots) enter the pores, while larger molecules (blue dots) bypass them, eluting faster due to their inability to penetrate the beads. - This separation based on **molecular size** is the principle of **size-exclusion chromatography** (also known as gel filtration or Sephadex chromatography), where Sephadex is a common matrix material. *Ion exchange chromatography* - This method separates molecules based on their **net charge**, using a stationary phase with charged groups that bind to oppositely charged molecules. - The image does not show any interaction based on charge; instead, it illustrates differences in how molecules navigate around or through the beads. *Gas liquid chromatography* - This technique separates volatile compounds based on their **differential partitioning** between a mobile gas phase and a stationary liquid phase coated on a solid support. - The illustration shows a liquid-phase column separation, not a gaseous mobile phase or vaporization of analytes. *Affinity chromatography* - This method separates molecules based on their **specific, reversible binding** to a ligand immobilized on the stationary phase. - The image does not indicate any specific binding interactions between the molecules being separated and the column matrix; rather, it shows physical exclusion based on size.

Q: The technique shown in the image is:

High performance liquid chromatography. ***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.

Q: Which is the correct sequence of steps in isolating desirable protein using recombinant DNA technology? 1. Expression of protein and lysis of the bacterial cell 2. Incorporation of genes into bacteria 3. SDS PAGE 4. Protein elution 5. Column chromatography

2,1,3,5,4. ***2,1,3,5,4*** - This sequence accurately reflects the typical order of operations in **recombinant protein isolation**: first, the gene is introduced into bacteria, then protein is expressed and cells lysed, followed by **SDS-PAGE as an intermediate quality check** to confirm protein expression before proceeding to purification steps (column chromatography and elution). - The process starts with gene incorporation, includes an analytical checkpoint after lysis, and ends with purified protein elution. *2,4,5,3,1* - This sequence is incorrect because **protein elution (4)** and **column chromatography (5)** are purification steps that occur *after* protein expression and cell lysis. - **Lysis (1)** cannot happen after elution, as cells must be lysed first to release the protein for purification. *1,2,4,3,5* - This sequence is incorrect because **expression and lysis (1)** must occur *after* the gene has been **incorporated into bacteria (2)** - the gene must be present before it can be expressed. - Additionally, **protein elution (4)** should follow **column chromatography (5)**, as elution is the step where protein is collected from the chromatography column. *1,5,2,4,3* - This sequence is incorrect because **incorporation of genes (2)** must be the first step - the gene needs to be in the bacteria before any expression, lysis, or purification can occur. - Starting with **expression and lysis (1)** before gene incorporation is impossible.

Q: Which of the following methods cannot be used to precipitate proteins?

Moving pH away from isoelectric pH. ***Moving pH away from isoelectric pH*** - Proteins are **least soluble** at their **isoelectric point (pI)**, where their net charge is zero, causing them to aggregate and precipitate. - Moving the pH **away from the isoelectric point** increases the net charge on the protein, enhancing its solubility and preventing precipitation. *Add alcohol and acetone* - **Organic solvents** like alcohol and acetone reduce the dielectric constant of water, weakening the **hydrophobic interactions** that maintain protein solubility. - This leads to increased protein-protein interactions and **precipitation** as the protein unfolds or aggregates. *Using heavy metal ions* - **Heavy metal ions** (e.g., lead, mercury) are positively charged and bind strongly to the negatively charged groups on proteins, such as **carboxylates** and **sulfhydryl groups**. - This binding can disrupt protein structure, lead to aggregation, and cause **precipitation**. *Adding trichloroacetic acid* - **Trichloroacetic acid (TCA)** is a strong acid that significantly lowers the pH of the solution, causing proteins to become **protonated**. - This change in charge and the disruption of **salt bridges** and hydrogen bonds lead to protein denaturation and **precipitation**.

Q: Glycated hemoglobin (HbA1c) is best measured using?

Ion exchange chromatography. ***Ion exchange chromatography*** - This method separates hemoglobin variants based on their **charge differences** due to the glucose molecule attached to HbA1c. - It is a highly sensitive and specific method for quantifying HbA1c, widely used in clinical laboratories. *Isoelectric focusing* - This technique separates molecules based on their **isoelectric point (pI)**, the pH at which they have no net charge. - While it can differentiate some hemoglobin variants, it is generally **less efficient and more complex** for routine HbA1c measurement compared to ion exchange chromatography. *Affinity chromatography* - This method separates molecules based on their **specific binding affinity** to a ligand immobilized on a stationary phase. - While it has been explored for HbA1c measurement, it is **not the most commonly used** or preferred method due to potential interferences and cost compared to ion exchange chromatography. *Electrophoresis* - This technique separates molecules based on their **charge and size** in an electric field. - While it can separate major hemoglobin variants, it has **lower resolution and accuracy** for routine HbA1c quantification compared to more specialized chromatographic methods, making it less ideal for precise measurement.

Q: Western blot is done for:

Protein. ***Protein*** - **Western blot** is a laboratory technique used to detect specific **proteins** in a sample of tissue homogenate or extract. - It involves separating proteins by **electrophoresis**, transferring them to a membrane, and then detecting them using **antibodies**. *RNA* - **Northern blot** is the technique specifically designed for the detection and analysis of **RNA** molecules. - It involves separating RNA fragments by **electrophoresis**, transferring them to a membrane, and querying with a labeled probe. *Lipid* - There is no direct "lipid blot" technique analogous to Western, Northern, or Southern blots. - **Lipids** are typically analyzed using techniques such as mass spectrometry, thin-layer chromatography, or gas chromatography. *DNA* - **Southern blot** is the molecular biology method used for the detection of specific **DNA** sequences in DNA samples. - It involves fragmenting DNA, separating by **electrophoresis**, and then hybridizing with a labeled DNA probe.

Q: Western blot is used for:

Proteins. ***Proteins*** - **Western blot** (also known as protein immunoblot) is a widely used analytical technique in molecular biology and immunogenetics to **detect specific proteins** in a given sample. - It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and then detecting the protein of interest using specific **antibodies**. *RNA* - The technique used to detect RNA is called **Northern blot**, which involves separating RNA fragments by size and then detecting specific RNA sequences using nucleic acid probes. - While both Northern and Western blots involve electrophoresis and transfer to a membrane, the target molecule is different. *Maternal DNA* - Detection of specific DNA sequences, including maternal DNA or fetal DNA, is typically performed using techniques like **Southern blot** or, more commonly now, **PCR-based methods** and **next-generation sequencing**. - Maternal DNA itself is not the specific target of a Western blot; proteins derived from any source of DNA, maternal or otherwise, would be the target. *DNA* - The technique primarily used for the detection of specific DNA sequences is **Southern blot**, which involves separating DNA fragments by size and using labeled probes to identify target sequences. - Western blot is fundamentally designed for protein analysis, relying on antibody-antigen recognition rather than DNA hybridization.

Q: Northern blot technique is used in the detection of:

RNA. ***RNA*** - The **Northern blot** technique specifically detects and measures **RNA** sequences in a sample - It involves separating RNA fragments by **gel electrophoresis**, transferring them to a membrane, and hybridizing with a **labeled complementary probe** - **Memory aid:** SNoW DRoP - **S**outhern = D**NA**, **No**rthern = **R**NA, **W**estern = **P**rotein *Incorrect: Plasmid* - **Plasmids** are circular **DNA** molecules, not RNA - Their detection uses **agarose gel electrophoresis** with staining, or **Southern blotting** for specific DNA sequence identification - Northern blot is designed specifically for RNA, not DNA-based structures *Incorrect: Prion* - **Prions** are **misfolded proteins** causing neurodegenerative diseases (e.g., Creutzfeldt-Jakob disease) - They are **not nucleic acids** (neither DNA nor RNA) - Detection requires **Western blot** or immunohistochemistry for protein analysis *Incorrect: DNA* - Detection of specific **DNA** sequences uses the **Southern blot** technique - Southern blot employs DNA probes to hybridize with target DNA after gel electrophoresis - Northern blot is exclusively for RNA, not DNA

Q: Spectroscopy primarily studies the interaction between _____ and matter.

Electromagnetic radiation. ***Electromagnetic radiation*** - **Spectroscopy** is the study of the interaction between **matter** and **electromagnetic radiation**, which includes a range from radio waves to gamma rays. - This interaction provides information about the **structure, composition, and physical properties** of matter. *Alpha particles* - **Alpha particles** are composed of two protons and two neutrons, essentially a helium nucleus. - While they interact with matter, their study is more characteristic of **nuclear physics** and **radioactivity**, not the primary focus of spectroscopy. *Protons* - **Protons** are subatomic particles with a positive charge found in the nucleus of an atom. - Their interaction with matter is studied in various fields, but spectroscopy primarily uses **electromagnetic radiation** to probe atomic and molecular energy levels. *Positrons* - **Positrons** are the antimatter counterparts of electrons, having a positive charge. - Their interaction with matter leads to **annihilation events** producing gamma rays, which is fundamental to techniques like Positron Emission Tomography (**PET**), but not the broad field of spectroscopy.

Q: Substance with same atomic number but different mass number –

Isotope. ***Isotope*** - **Isotopes** are atoms of the same element, meaning they have the **same atomic number** (number of protons). - They differ in their **mass number** due to varying numbers of neutrons. *Mineral* - A **mineral** is a naturally occurring solid, inorganic substance with a definite chemical composition and crystal structure. - This definition does not relate to the atomic or mass numbers of individual atoms. *Isobar* - **Isobars** are atoms of different elements that have the **same mass number** but different atomic numbers. - This is the opposite of the given description. *Atom* - An **atom** is the basic unit of a chemical element. - While isotopes are types of atoms, the term "atom" itself is too broad and doesn't specify the relationship described.

Question 1

The following method of chromatography is called:

Accepted Answer

Sephadex chromatography

Answer

Ion exchange chromatography

Answer

Gas liquid chromatography

Answer

Affinity chromatography

Question 2

The technique shown in the image is:

Accepted Answer

High performance liquid chromatography

Answer

Haemoglobin electrophoresis

Answer

Gel electrophoresis

Answer

Tandem mass spectrometry

Question 3

Which is the correct sequence of steps in isolating desirable protein using recombinant DNA technology?

1. Expression of protein and lysis of the bacterial cell
2. Incorporation of genes into bacteria
3. SDS PAGE
4. Protein elution
5. Column chromatography

Accepted Answer

2,1,3,5,4

Answer

2,4,5,3,1

Answer

1,2,4,3,5

Answer

1,5,2,4,3

Question 4

Which of the following methods cannot be used to precipitate proteins?

Accepted Answer

Moving pH away from isoelectric pH

Answer

Add alcohol and acetone

Answer

Using heavy metal ions

Answer

Adding trichloroacetic acid

Question 5

Glycated hemoglobin (HbA1c) is best measured using?

Accepted Answer

Ion exchange chromatography

Answer

Isoelectric focusing

Answer

Affinity chromatography

Answer

Electrophoresis

Question 6

Western blot is done for:

Accepted Answer

Protein

Answer

RNA

Answer

Lipid

Answer

DNA

Question 7

Western blot is used for:

Accepted Answer

Proteins

Answer

RNA

Answer

Maternal DNA

Answer

DNA

Question 8

Northern blot technique is used in the detection of:

Accepted Answer

RNA

Answer

Plasmid

Answer

Prion

Answer

DNA

Question 9

Spectroscopy primarily studies the interaction between _____ and matter.

Accepted Answer

Electromagnetic radiation

Answer

Alpha particles

Answer

Protons

Answer

Positrons

Question 10

Substance with same atomic number but different mass number –

Accepted Answer

Isotope

Answer

Mineral

Answer

Isobar

Answer

Atom

Biochemical Techniques — MCQs

Biochemical Techniques — MCQs

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