Laboratory Biochemistry & Cell Biology

LIFE 212

Why must we understand proteins to understand cells?

Proteins are key molecules that govern cellular behavior.

What does "in vitro" mean in protein studies?

Studying proteins outside of cells, "in glass".

Do proteins usually act alone?

No, they function in concert with other molecules like proteins, DNA, or RNA.

What can proteins be combined with in vitro?

Other molecules to measure function and behavior.

What are plasmids?

"Mini chromosomes" that encode proteins of interest.

How can plasmids be engineered?

To express a protein of interest.

What critical sequences do plasmids contain?

Replication origin, restriction enzyme sites (MCS), antibiotic resistance gene.

What is the function of the replication origin in a plasmid?

Allows independent copying in a bacterial cell.

What is the function of restriction enzyme sites in a plasmid?

Enable opening the plasmid to insert foreign DNA fragments.

What is the function of the antibiotic resistance gene in a plasmid?

Allows selection of bacteria that contain the plasmid.

How can plasmids be introduced into cells?

Bacteria: transformation; Eukaryotes: transformation (yeast) or transfection (mammals).

What happens after cells are transformed with a plasmid?

Cells grow and express genes encoded in the plasmid.

How is protein harvested from cells?

Cells are centrifuged, lysed to release contents, and protein is isolated.

What is an affinity tag?

A small protein region with high affinity for something useful to purify the protein.

How are affinity tags encoded?

In tandem with the protein of interest in the plasmid DNA.

Where can affinity tags be attached?

N-terminus or C-terminus of the protein.

How are proteins purified using affinity tags?

Beads bind the tag, lysate is removed, and protein is eluted from the beads.

How is a protein eluted from beads?

By using a molecule that competes with the bead binding.

What can fluorescent protein tags do?

Allow visualization of proteins by microscopy.

What does SDS-PAGE assess?

Abundance of protein and protein mass.

How does SDS-PAGE work?

Proteins form negatively charged SDS complexes and migrate through a polyacrylamide gel.

Does SDS-PAGE separate proteins based on shape or charge?

No, only based on mass.

What is used to visualize protein bands in SDS-PAGE?

A stain, e.g., coomassie.

What does the intensity of a protein band indicate?

Protein abundance.

How is SDS-PAGE used in protein purification?

To monitor the success of purification.

What does size exclusion chromatography (SEC) separate proteins by?

Size.

How do porous beads in SEC affect small proteins?

Small proteins pass through pores, increasing their path length.

How do large protein complexes behave in SEC?

Large proteins go around the beads and elute faster.

What can SEC be used to assess besides size?

Protein-protein interactions.

Which methods can directly visualize proteins?

X-ray crystallography and cryogenic electron microscopy (cryo-EM).

How are proteins oriented in cryo-EM samples?

Randomly in ice.

How are images processed in cryo-EM?

Algorithms sort molecules into orientation sets, then thousands of images are combined to generate high-resolution 3D structures.

What do modern biochemical techniques permit scientists to assess?

Protein behavior outside of a cell and interactions with other proteins.

How can protein structure be directly visualized?

By cryo-electron microscopy (cryo-EM).

What questions can in vitro studies help answer?

If protein X is sufficient to perform a certain behavior, or how protein X affects protein Y.

How do we know anything about cells?

By using model systems and organisms (yeast, flies, cultured human cells) and modern laboratory techniques.

What advantages do modern microscopy techniques provide?

High spatial and temporal resolution of cellular processes.

Why are non-human model systems useful for research?

They have short life cycles, are easy to engineer, and allow study of normal and disease states.

Why are model systems easier to maintain than animals?

Short generation/doubling time and amenable to genetic engineering.

How do model systems help study cellular processes?

By permitting manipulation of genes and observation of conserved biological pathways.

Why is yeast a valuable model system?

Short life cycles, easy to manipulate genes, and conservation of eukaryotic biological and biochemical pathways.

Which protein is an example of strong conservation across evolution?

Dynein.

Why is C. elegans an excellent model for developmental biology?

Transparent, easy to culture, fixed number of cells (959), and fully mapped cell lineage.

How can cultured human cells be studied?

In isolation in culture as a "human" model.

What are induced pluripotent stem cells (iPSCs)?

Pluripotent stem cells generated from adult cells that can differentiate into almost any cell type.

Why are iPSCs useful?

For drug discovery, disease analysis, and treatment.

What is one method to study protein function by reducing its levels?

Gene deletion or depletion by RNA interference (RNAi).

How does RNA interference (RNAi) work?

It reduces levels of an RNA of interest via binding of a small interfering RNA (siRNA).

What technology is used to delete or mutate a gene of interest?

CRISPR-Cas9.

How does CRISPR-Cas9 target a specific gene?

Scientists design guide RNA (gRNA) to direct Cas9 to the target gene.

What is Cas9 and what does it do?

An endonuclease that cuts DNA, producing a double-stranded break in the target gene.

How does Cas9-mediated cleavage lead to gene deletion or mutation?

Cells repair the break, often introducing mistakes, or donor DNA with mutations can be supplied.

How can protein localization and dynamics be studied?

By expressing fluorescent protein fusions (e.g., GFP) in cells.

What questions can fluorescent protein fusions answer?

Where the protein goes, when it goes there, and its dynamics inside the cell.

How can depletion and expression strategies be combined?

Deplete protein X via siRNA, then express a mutant variant to test if it rescues function.

What does "rescue" of protein function mean in experiments?

The mutant protein restores the lost function of the depleted or deleted protein.