Protein Isolation and Quantification Flashcards

A biotechnician needs to purify a large batch of a recombinant protein that has been engineered to include a polyhistidine tag (His-tag). Which type of chromatography would be the MOST effective and specific method for this initial capture step?

Answer: Immobilized Metal Affinity Chromatography (IMAC)

Immobilized Metal Affinity Chromatography (IMAC) is the most specific and effective method for purifying His-tagged proteins. The resin in the column is chelated with divalent metal ions (like Nickel or Cobalt) that have a high affinity for the series of histidine residues on the tag, allowing the target protein to bind tightly while most other cellular proteins wash through.

When performing a Bradford assay to create a standard curve, a technician observes that the color of the solution changes from brown to blue upon addition of the protein standards. What is the spectrophotometer measuring at 595 nm?

Answer: The absorbance of the protein-dye complex, where the dye has shifted to its blue form.

The Bradford assay is based on the binding of Coomassie Brilliant Blue G-250 dye to proteins. In its unbound, acidic state, the dye is reddish-brown with an absorbance maximum around 465 nm. When it binds to proteins, it converts to a stable blue form, causing the absorbance maximum to shift to 595 nm. The increase in absorbance at 595 nm is proportional to the amount of protein present.

In SDS-PAGE, what is the primary function of the sodium dodecyl sulfate (SDS) component in the sample loading buffer and the gel running buffer?

Answer: To denature proteins and impart a uniform negative charge-to-mass ratio.

SDS is an anionic detergent that serves two key functions: it disrupts the non-covalent bonds that maintain a protein's secondary and tertiary structure (denaturation), and it coats the linearized protein with a uniform negative charge. This ensures that during electrophoresis, the proteins migrate through the polyacrylamide gel based primarily on their molecular weight, not their intrinsic charge or shape.

A technician is performing size-exclusion chromatography (SEC) to separate a mixture of three proteins with different molecular weights: Protein A (150 kDa), Protein B (75 kDa), and Protein C (25 kDa). In what order will these proteins elute from the column?

Answer: Protein A, then B, then C

In size-exclusion chromatography, molecules are separated based on their size. The chromatography column is packed with porous beads. Larger molecules, like Protein A (150 kDa), are too big to enter the pores and thus travel around the beads, taking a shorter path and eluting first. Smaller molecules, like Protein C (25 kDa), can enter the pores, taking a longer, more convoluted path through the column, and therefore elute last.

A lab needs to accurately quantify the total protein concentration in crude cell lysates that contain a high concentration of detergents, such as Triton X-100. Which of the following quantification methods is generally more suitable and less prone to interference from these detergents?

Answer: Bicinchoninic acid (BCA) assay

The BCA assay is known for its compatibility with samples containing detergents up to 5%. Its chemistry, involving the reduction of copper ions, is less affected by common non-ionic and ionic detergents found in cell lysis buffers compared to the Bradford assay, which can show significant interference as detergents disrupt the dye-protein binding mechanism.

A biotechnician uses a spectrophotometer to measure the concentration of a purified protein by reading the absorbance at 280 nm (A280). This direct measurement method relies on the presence of which specific components within the protein?

Answer: Aromatic amino acids like tryptophan and tyrosine

The basis for direct UV protein quantification at 280 nm is the absorbance of light by the aromatic side chains of specific amino acids, primarily tryptophan and tyrosine, and to a lesser extent, cysteine-cysteine disulfide bonds. Proteins lacking these residues cannot be quantified by this method.