Biochemical Techniques Lab 2: Protein Precipitation

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Questions and Answers

What is the primary purpose of protein precipitation?

To eliminate interferences

To purify proteins

To study protein folding

All of the above (correct)

What is the isoelectric point (pI) of a protein?

The pH at which a protein is most soluble

The pH at which a protein carries zero net electrical charge (correct)

The pH at which a protein is most reactive

The pH at which a protein is least soluble

What happens to the protein when acids are added to the solution in acid precipitation?

It becomes negatively charged

It becomes positively charged (correct)

It breaks down into smaller molecules

It remains neutral

What is the most popular acid used in acid precipitation?

Trichloroacetic Acid (TCA) Signup and view all the answers

Why is it important to run experiments at low temperatures in alcohol precipitation?

To preserve the biological functions of proteins Signup and view all the answers

What is the effect of adding alcohol to the solution in alcohol precipitation?

It reduces the hydration of the protein Signup and view all the answers

What is the role of the hydration shell in protein folding and function?

It plays a very important role Signup and view all the answers

What is the purpose of the hydration shell around proteins?

To surround proteins with water molecules Signup and view all the answers

What is the effect of adding low salt concentrations to a protein solution?

Protein solubility increases Signup and view all the answers

What is the purpose of adding high salt concentrations to a protein solution?

To precipitate proteins Signup and view all the answers

What is the advantage of using (NH4)2SO4 in salting-out technique?

Its high solubility and lack of buffering capacity Signup and view all the answers

What determines the effective salt concentration for protein precipitation?

The number and position of polar groups, molecular weight, pH, and temperature Signup and view all the answers

What is a disadvantage of the salting-out technique?

A high amount of salt must be removed from the precipitate Signup and view all the answers

What method is used to remove salt from the protein sample?

Dialysis or gel filtration chromatography Signup and view all the answers

Why do higher molecular weight proteins precipitate out at lower salt concentrations?

Because they have a lower solubility Signup and view all the answers

What is the term for the process where salt ions attract the protein's functional groups?

Salting in Signup and view all the answers

Study Notes

Protein Precipitation

Protein precipitation separates proteins from a solution to eliminate interferences or purify them.
The efficacy of precipitation methods depends on the solubility and molecular structure of the protein.

Acid Precipitation

Acid precipitation uses changes in pH to precipitate proteins.
Each protein has a specific pH, called isoelectric point (pI), where the molecule carries zero net electrical charge.
At the isoelectric point, the molecule is least soluble in water and tends to precipitate or aggregate.
Adding acids, such as TCA (Trichloroacetic Acid), reduces pH, making the protein positively charged, disrupting water molecules, and causing precipitation.

Alcohol Precipitation

Organic solvents, like ethanol, methanol, and acetone, can be used to precipitate proteins.
Adding alcohol reduces hydration of the protein, leading to aggregation and precipitation.
Experiments should be run at low temperatures to preserve biological functions.

Salting Precipitation

Low salt concentrations increase protein solubility through "salting in," where salt ions attract functional groups on the protein.
High salt concentrations limit water molecules available for protein interaction, leading to precipitation through "salting out."
Zinc sulfate and ammonium sulfate are examples of salts used in this method.
Salting precipitation is a simple, rapid, and non-harmful method for protein precipitation.
Higher molecular weight proteins precipitate out at lower salt concentrations.
(NH4)2SO4 is a commonly used salt due to its high solubility, lack of buffering capacity, low cost, and low density of the resulting solution.
Critical factors affecting effective salt concentration include:
- Number and position of polar groups
- Molecular weight of the protein
- pH of the solution
- Temperature of precipitation

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Description

This quiz covers the principles and methods of protein precipitation in biochemical laboratories, including the importance of hydration shell and solubility in protein separation.