Biochemical Techniques Practice Questions

Q: Separation of proteins based on size is done by

SDS-Polyacrylamide gel electrophoresis. ***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.

Q: Which technique is used for protein separation based on molecular size?

Gel filtration chromatography. ***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.

Q: Which among the following is identified by Western blotting

Proteins. ***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.

Q: Which of the following methods of protein separation is not dependent on molecular size?

Ion-exchange chromatography. ***Ion-exchange chromatography*** - This method separates proteins based on their **net charge** at a specific pH. - Proteins bind to a charged resin based on their charge, independent of their size. *Gel filtration chromatography* - Separates proteins based on their **molecular size** and shape as they pass through a porous matrix. - **Larger molecules** elute first as they cannot enter the pores, while smaller molecules are retained. *SDS-PAGE* - **Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis** separates proteins primarily based on their **molecular weight**. - SDS denatures proteins and confers a uniform negative charge, allowing migration through a gel matrix based on size. *Ultracentrifugation* - Separates macromolecules and particles based on their **sedimentation rate**, which is influenced by **molecular mass**, density, and shape. - While molecular size is a factor, density and shape also play significant roles in the separation process.

Q: Which of the following defines isotopes?

Same atomic number and different mass number. ***Same atomic number and different mass number*** - Isotopes are atoms of the same element, meaning they have the same number of **protons**, which defines the **atomic number**. - They differ in the number of **neutrons**, leading to a different **mass number** (protons + neutrons). *Different atomic number and same mass number* - This describes **isobars**, which are atoms of different elements but have the same total number of nucleons. - Having a different **atomic number** means they are different elements. *Same atomic number and same mass number* - This describes identical atoms of the **same element**, as both the number of protons and neutrons are the same. - These are simply different atoms of the same nuclide. *Different atomic number and different mass number* - This describes **different elements** with different numbers of protons and neutrons. - There is no specific term for this general category, as it just refers to distinct atomic species.

Q: All the following statements about hybridoma technology are true except:

Myeloma cells in salvage pathway grow well in HAT medium.. ***Myeloma cells in salvage pathway grow well in HAT medium.*** - Myeloma cells used in hybridoma technology are typically deficient in **HGPRT (hypoxanthine-guanine phosphoribosyltransferase)**, which is essential for the **salvage pathway** of nucleotide synthesis. - Such HGPRT-deficient myeloma cells **cannot survive** in **HAT medium** because aminopterin blocks the de novo pathway, and without a functional salvage pathway, they cannot synthesize DNA. - This is the basis of **HAT selection** - only hybridoma cells (fused B cells + myeloma cells) survive because B cells provide functional HGPRT. *HGPRT is required for the salvage pathway.* - **HGPRT** (hypoxanthine-guanine phosphoribosyltransferase) is crucial for the **purine salvage pathway**, enabling cells to reuse hypoxanthine and guanine for nucleotide synthesis. - Cells with functional HGPRT can survive in HAT medium by using the salvage pathway when de novo synthesis is blocked. *Aminopterin, a folate antagonist, inhibits de novo pathway.* - **Aminopterin** is indeed a **folate antagonist** that inhibits **dihydrofolate reductase**, an enzyme critical for the *de novo* synthesis of purines and thymidylate. - By blocking this enzyme, aminopterin effectively **shuts down the *de novo* nucleotide synthesis pathway**, forcing cells to rely on the salvage pathway. *Specific antibody-producing cells are integrated with myeloma cells.* - In hybridoma technology, **antibody-producing B lymphocytes** (plasma cells) are fused with **myeloma cells** to create hybrid cells called hybridomas. - This fusion combines the antibody specificity and production capacity of B cells with the immortality and rapid division of myeloma cells, allowing for **continuous production** of monoclonal antibodies.

Q: Which amino acid migrates fastest on chromatography on carboxymethyl cellulose medium?

Aspartic acid. ***Aspartic acid*** - Carboxymethyl cellulose (CMC) is a **cation exchange resin** with negatively charged carboxyl groups (-COO⁻). It binds positively charged molecules while **repelling negatively charged molecules**. - **Aspartic acid** is an **acidic amino acid** with a net negative charge at physiological pH, causing it to be **repelled** by the negatively charged resin and migrate **fastest**. - This electrostatic repulsion prevents binding and allows rapid elution. *Glycine* - **Glycine** is a **neutral amino acid** with minimal net charge at physiological pH. - It has weak or no electrostatic interaction with the resin, making it migrate at a moderate speed. - It lacks ionizable side chains, so its migration is slower than negatively charged amino acids but faster than positively charged ones. *Lysine* - **Lysine** is a **basic amino acid** and carries a net positive charge at physiological pH. - It **binds strongly** to the negatively charged CMC resin through electrostatic attraction. - This strong binding causes it to migrate **very slowly** or remain bound until eluted with high salt concentration. *Valine* - **Valine** is a **neutral, nonpolar amino acid** with no significant charge at physiological pH. - It has minimal electrostatic interaction with the CMC resin and migrates at a moderate speed. - It moves slower than negatively charged amino acids due to lack of electrostatic repulsion from the resin.

Q: Nephelometry measures the light?

Scattered. ***Scattered*** - **Nephelometry** measures the **light scattered** by particles in a liquid sample. - The intensity of the scattered light is directly proportional to the concentration of the analyte, such as immune complexes or proteins. *Refracted* - **Refraction** is the **bending of light** as it passes from one medium to another, like through a lens, and is not the principle behind nephelometry. - Refractive index measurements are used in techniques like refractometry, not nephelometry. *Transmitted* - **Transmitted light** is the light that passes directly through a sample without being absorbed or scattered. - Measuring transmitted light is the basis of **turbidimetry**, a related but distinct technique. *Absorbed* - **Absorbed light** is the light that is taken up by a substance, preventing it from passing through or being scattered. - Measuring absorbed light is the principle of **spectrophotometry** (e.g., colorimetry), not nephelometry.

Q: Pulsed gel electrophoresis is used for:

DNA. ***DNA*** - **Pulsed-field gel electrophoresis (PFGE)** is a specialized technique used to separate **large DNA molecules** that are difficult to resolve using conventional gel electrophoresis. - It involves periodically changing the direction of the electric field, which forces large DNA molecules to reorient themselves, allowing for better separation based on size up to **10 Mb**. *RNA* - While conventional gel electrophoresis (e.g., agarose or polyacrylamide gels) can be used to separate **RNA molecules**, PFGE is not typically employed for RNA. - RNA molecules are generally much smaller than the large DNA fragments for which PFGE is designed, and their secondary structures can interfere with pulsed-field separation. *Protein* - **Proteins** are separated using different types of electrophoresis, such as **SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)**, which separates proteins primarily by size. - PFGE is specifically designed for nucleic acids, particularly very large DNA, and is not suitable for protein separation. *Ribosome* - **Ribosomes** are large macromolecular complexes composed of ribosomal RNA and proteins. - Techniques like **sucrose gradient centrifugation** or specialized gel electrophoresis (e.g., non-denaturing agarose gels for intact ribosomes) are used to separate ribosomes, not PFGE.

Q: For DNA extraction from blood samples, the preferred anticoagulant is:

EDTA. ***EDTA*** - **EDTA** (ethylenediaminetetraacetic acid) acts as an anticoagulant by **chelating calcium ions**, which are essential for the coagulation cascade, making it ideal for DNA extraction. - Using an EDTA collection tube ensures that the blood sample remains in its liquid state, preventing clot formation which can trap DNA and make isolation difficult. *Plain bulb* - A plain bulb refers to a tube without any anticoagulant, allowing the blood to **clot naturally**. - While serum can be obtained from such a tube, the DNA would be entrapped within the clot, making its extraction **less efficient and potentially damaging**. *Formalin* - **Formalin** (a solution of formaldehyde) is a fixative used to preserve tissue morphology by **cross-linking proteins**. - While useful for histopathology, it **damages DNA** through chemical modifications and fragmentation, making it unsuitable for DNA isolation or genetic analysis. *None of the options* - This option is incorrect because **EDTA is a widely recognized and appropriate** anticoagulant for preserving DNA samples from blood for molecular studies.

Question 1

Separation of proteins based on size is done by

Accepted Answer

SDS-Polyacrylamide gel electrophoresis

Answer

Affinity chromatography

Answer

High performance liquid chromatography

Answer

Ion exchange chromatography

Question 2

Which technique is used for protein separation based on molecular size?

Accepted Answer

Gel filtration chromatography

Answer

Affinity chromatography

Answer

HPLC

Answer

Salting out

Question 3

Which among the following is identified by Western blotting

Accepted Answer

Proteins

Answer

t-RNA

Answer

RNA

Answer

DNA

Question 4

Which of the following methods of protein separation is not dependent on molecular size?

Accepted Answer

Ion-exchange chromatography

Answer

Gel filtration chromatography

Answer

SDS-PAGE

Answer

Ultracentrifugation

Question 5

Which of the following defines isotopes?

Accepted Answer

Same atomic number and different mass number

Answer

Different atomic number and same mass number

Answer

Same atomic number and same mass number

Answer

Different atomic number and different mass number

Question 6

All the following statements about hybridoma technology are true except:

Accepted Answer

Myeloma cells in salvage pathway grow well in HAT medium.

Answer

HGPRT is required for the salvage pathway.

Answer

Aminopterin, a folate antagonist, inhibits de novo pathway.

Answer

Specific antibody-producing cells are integrated with myeloma cells.

Question 7

Which amino acid migrates fastest on chromatography on carboxymethyl cellulose medium?

Accepted Answer

Aspartic acid

Answer

Glycine

Answer

Lysine

Answer

Valine

Question 8

Nephelometry measures the light?

Accepted Answer

Scattered

Answer

Refracted

Answer

Transmitted

Answer

Absorbed

Question 9

Pulsed gel electrophoresis is used for:

Accepted Answer

DNA

Answer

RNA

Answer

Protein

Answer

Ribosome

Question 10

For DNA extraction from blood samples, the preferred anticoagulant is:

Accepted Answer

EDTA

Answer

Plain bulb

Answer

Formalin

Answer

None of the options

Biochemical Techniques — MCQs

Biochemical Techniques — MCQs

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