Protein precipitation technique MCQs With Answer

Introduction: Protein precipitation is a fundamental sample-preparation technique in bioanalytical workflows, especially for M.Pharm students learning modern methods for drug analysis and proteomics. This blog presents focused multiple-choice questions covering principles, common precipitants (organic solvents, acids, salts), procedural parameters (solvent ratios, temperature, centrifugation), and practical challenges such as co-precipitation, matrix effects, and downstream assay compatibility. The questions emphasize decision-making for LC‑MS/MS and proteomic workflows, troubleshooting poor recoveries, and selecting appropriate cleaning strategies. Use these MCQs to test and strengthen your understanding of how protein precipitation affects analyte recovery, assay interference, and sample cleanliness in regulated bioanalytical settings.

Q1. What is the primary mechanism of “salting-out” protein precipitation?

• Salt ions stabilize protein tertiary structure increasing solubility
• Salt ions compete for water, reducing protein solvation and causing aggregation
• Salt ions chemically modify amino acids leading to covalent cross-linking
• Salt ions increase the dielectric constant to keep proteins in solution

Correct Answer: Salt ions compete for water, reducing protein solvation and causing aggregation

Q2. Which pair of organic solvents is most commonly used for protein precipitation in plasma sample preparation for LC‑MS/MS?

• Chloroform and hexane
• Acetonitrile and methanol
• Water and glycerol
• Ethyl acetate and dichloromethane

Correct Answer: Acetonitrile and methanol

Q3. Which precipitating agents are typically used to precipitate proteins by strong acidification?

• Sodium chloride (NaCl) or potassium chloride (KCl)
• Trichloroacetic acid (TCA) or perchloric acid (PCA)
• Ammonium sulfate or sodium sulfate
• Acetone or isopropanol

Correct Answer: Trichloroacetic acid (TCA) or perchloric acid (PCA)

Q4. For routine plasma protein precipitation prior to LC‑MS, a commonly used organic solvent-to-plasma volume ratio is:

• 1:1 (solvent : plasma)
• 2:1 (solvent : plasma)
• 3:1 (solvent : plasma)
• 10:1 (solvent : plasma)

Correct Answer: 3:1 (solvent : plasma)

Q5. What is the main benefit of performing protein precipitation at 0–4°C?

• Speeds up enzymatic degradation to clear proteins faster
• Enhances precipitation efficiency and reduces proteolytic activity
• Prevents phase separation of organic solvents
• Causes proteins to re-solubilize into the supernatant

Correct Answer: Enhances precipitation efficiency and reduces proteolytic activity

Q6. A common disadvantage of trichloroacetic acid (TCA) precipitation is:

• It leaves no pellet and cannot concentrate protein
• It produces a pellet that is easy to re-dissolve without modification
• It can cause irreversible denaturation and is difficult to re-solubilize
• It is ineffective at precipitating proteins from serum

Correct Answer: It can cause irreversible denaturation and is difficult to re-solubilize

Q7. To reduce phospholipid-related matrix effects prior to LC‑MS, which sample cleanup strategy is generally most effective?

• Simple protein precipitation without additional steps
• Solid-phase extraction (SPE)
• Heating the sample to 60°C
• Diluting the sample tenfold with water

Correct Answer: Solid-phase extraction (SPE)

Q8. Which salt is most frequently used for classical “salting-out” precipitation due to its strong salting-out power?

• Sodium chloride (NaCl)
• Potassium chloride (KCl)
• Magnesium sulfate (MgSO4)
• Ammonium sulfate ((NH4)2SO4)

Correct Answer: Ammonium sulfate ((NH4)2SO4)

Q9. After adding precipitating solvent and incubating, the standard laboratory step to separate precipitated protein from supernatant is:

• Centrifugation at appropriate speed and temperature
• Dialysis against distilled water
• Direct injection without separation
• Vortexing continuously for 60 minutes

Correct Answer: Centrifugation at appropriate speed and temperature

Q10. A major cause of low analyte recovery after protein precipitation is:

• Complete dissolution of all proteins into the supernatant
• Co-precipitation of analyte bound to proteins
• Over-dilution of the supernatant
• Excessive heating of the supernatant

Correct Answer: Co-precipitation of analyte bound to proteins

Q11. Precipitation of a protein at its isoelectric point (pI) occurs because:

• The protein has maximal net charge and repels other molecules
• The protein carries no net charge, reducing electrostatic repulsion and solubility
• The protein becomes more hydrated and soluble
• The protein forms covalent bonds with buffer ions

Correct Answer: The protein carries no net charge, reducing electrostatic repulsion and solubility

Q12. For proteomics workflows aiming to collect precipitated intact proteins for subsequent digestion, which precipitant is commonly preferred?

• Cold acetone precipitation
• Room-temperature acetonitrile precipitation
• Heating with 1% SDS
• Direct injection without cleanup

Correct Answer: Cold acetone precipitation

Q13. Which precipitating agent is most likely to interfere with a Bradford protein assay if residual reagent remains?

• Acetone
• Trichloroacetic acid (TCA)
• Ammonium sulfate
• Cold ethanol

Correct Answer: Trichloroacetic acid (TCA)

Q14. Typical centrifugation conditions to pellet precipitated proteins after solvent addition for analytical workflows are:

• 500 x g for 1 minute at room temperature
• 2,000 x g for 5 minutes at 37°C
• 10,000–15,000 x g for 10–15 minutes at 4°C
• 100,000 x g for 24 hours at room temperature

Correct Answer: 10,000–15,000 x g for 10–15 minutes at 4°C

Q15. In small-molecule bioanalysis after protein precipitation, the target analyte is typically found in:

• The protein pellet
• The supernatant
• The centrifuge tube wall only
• The discarded wash solvent exclusively

Correct Answer: The supernatant

Q16. Which statement about cold acetone precipitation is correct?

• It requires pre-chilled acetone and cold incubation to precipitate efficiently
• It is ineffective below 20°C
• Acetone stabilizes proteins in solution preventing aggregation
• It always leaves proteins fully soluble for direct injection

Correct Answer: It requires pre-chilled acetone and cold incubation to precipitate efficiently

Q17. To best compensate for losses during protein precipitation when quantifying drugs in plasma by LC‑MS, when should you add the internal standard (IS)?

• After centrifugation to the supernatant only
• Immediately before injection into LC
• Before protein precipitation to account for extraction losses
• Never add an internal standard for precipitation methods

Correct Answer: Before protein precipitation to account for extraction losses

Q18. Compared to methanol, acetonitrile as a precipitating solvent typically:

• Produces more viscous extracts and poorer protein removal
• Generally precipitates proteins more effectively and yields cleaner supernatants for LC‑MS
• Is less compatible with LC‑MS and rarely used
• Does not mix with plasma and forms an immiscible layer

Correct Answer: Generally precipitates proteins more effectively and yields cleaner supernatants for LC‑MS

Q19. What is the primary purpose of washing a protein pellet briefly with cold organic solvent after precipitation?

• To increase the amount of bound analyte in the pellet
• To remove residual salts, lipids, and small molecules while minimizing analyte loss
• To completely solubilize the pellet for direct injection
• To neutralize the pellet to pH 7.4

Correct Answer: To remove residual salts, lipids, and small molecules while minimizing analyte loss

Q20. “Co-precipitation” during protein precipitation refers to which problem, and how is it commonly minimized?

• Loss of solvent due to evaporation; minimized by sealing tubes
• Loss of analyte with the protein pellet; minimized by adding internal standard before precipitation and optimizing solvent ratio
• Formation of inorganic precipitates; minimized by adding acid
• Complete solubilization of proteins; minimized by heating

Correct Answer: Loss of analyte with the protein pellet; minimized by adding internal standard before precipitation and optimizing solvent ratio

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