Working with Proteins: Purification and Study

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What is one of the factors to consider when stabilizing isolated proteins?

Storing proteins at high temperatures

Blocking degradative enzymes such as nucleases & proteases (correct)

Reducing pH levels

Increasing adsorption to surface

Which technique is NOT used in protein purification according to the text?

Salting out

Electrophoresis

Chromatography

Ionization (correct)

What happens when salt is added during the 'salting in' process of protein solubility?

Protein solubility increases as salt is added (correct)

Additional ions intensify attractive forces between proteins

Proteins precipitate at lower salt concentrations

Solubility of protein decreases due to shielded charges

What is the most commonly used reagent for salting out proteins?

Ammonium sulfate Signup and view all the answers

How are proteins fractionated according to solubility in the text?

By molecular size Signup and view all the answers

Which enzyme-linked assay technique is mentioned in the text for assaying proteins?

ELISA Signup and view all the answers

What happens to the solubility of a protein when its net charge is zero?

It is least soluble Signup and view all the answers

In chromatography, what is the purpose of the mobile phase?

To be a liquid for fractionation Signup and view all the answers

Which type of chromatography separates molecules based on size and shape?

Gel filtration chromatography Signup and view all the answers

What type of matrix is frequently used in ion-exchange chromatography?

Cellulose and agarose-based resin Signup and view all the answers

How are proteins eluted from an ion exchanger in chromatography?

By using a buffer with higher salt concentration or pH that reduces affinity to matrix Signup and view all the answers

What is monitored in the column effluent during ion-exchange chromatography?

Absorbance spectroscopy at 280 nm Signup and view all the answers

What is the purpose of affinity chromatography?

To estimate the mass of unknown proteins Signup and view all the answers

In affinity chromatography, how are proteins separated from other substances?

By washing the column with a high concentration of free ligand Signup and view all the answers

What is the role of pH in electrophoresis of proteins?

To separate proteins based on their charges Signup and view all the answers

How are separated amino acids made visible in paper electrophoresis?

By coating the paper with ninhydrin Signup and view all the answers

What is the purpose of SDS-PAGE in protein separation?

To separate proteins according to molecular mass Signup and view all the answers

Why is Coomassie blue used in electrophoresis of proteins?

To visualize separated proteins bands Signup and view all the answers

How does SDS bind to proteins?

By hydrophobic interaction Signup and view all the answers

What property of proteins does SDS-PAGE estimate?

Molecular weight Signup and view all the answers

How are proteins separated in isoelectric focusing?

According to their isoelectric point Signup and view all the answers

What forms the pH gradient in isoelectric focusing?

Low molecular mass oligomers bearing amino and carboxylic acid groups Signup and view all the answers

In protein sequencing, how can the number of different subunits be determined?

By identifying the end group residues Signup and view all the answers

What does the equal amount of Glycine and Phenylalanine in insulin indicate?

Insulin contains two nonidentical polypeptide chains Signup and view all the answers

What is the key feature of using 1-fluoro-2,5-dinitrobenzene (FDNB) in protein sequencing for N-terminal identification?

Formation of a yellow color for easy visualization Signup and view all the answers

Which enzyme is typically used for C-terminal identification in protein sequencing?

Carboxypeptidase A Signup and view all the answers

What is the function of dansyl chloride in protein sequencing for N-terminal identification?

Intense yellow fluorescence for precise identification Signup and view all the answers

Which statement about carboxypeptidase A is correct?

Does not cleave C-terminal Arg or Lys residues Signup and view all the answers

How does aminopeptidase contribute to protein sequencing?

Liberates amino acids one at a time from the N-terminus Signup and view all the answers

What caution is provided regarding carboxypeptidase hydrolysis in protein sequencing?

Cleavage of the first residue slowly & the second quickly suggests two different polypeptides Signup and view all the answers