ABIO 301 EXAM #2

Western blot: what does it detect?

Size and amount of a specific protein in a cell extract

Western blot: Steps

SDS-PAGE gel -> transfer to nitrocellulose -> block (milk) -> antibody -> enzyme makes light -> detect band

Western blot: why SDS-PAGE first?

Separates proteins by size

Western blot: what is the blocking step for?

Prevents non-specific antibody binding (powdered milk is used)

Western blot: how is the signal made?

Antibody is attached to an enzyme that converts a colorless substrate into a light reaction (camera/film)

Western blot: is it quantitative?

Semi-quantitative (pixels in band compared to a standard curve)

Western blot: limitations

No info on where the protein is in the cell; only 1 protein at a time (3-4 max)

Western blot: another name

Protein immunoblot

Western blot: how does a fusion protein look?

Larger band than the wild type protein

SDS

Sodium dodecyl sulfate; harsh anionic detergent that unfolds/denatures proteins for size-based separation

Triton X-100

Non-ionic detergent; lyses cells/dissolves membranes while keeping proteins functional

SDS vs Triton X-100: which denatures proteins?

SDS (anionic). Triton X-100 (non-ionic) keeps proteins active

PAGE: what it stands for?

Polyacrylamide gel electrophoresis

PAGE: what does it do?

proteins move through a gel in an electric field and separate mainly by size

Antibody (immunoglobulin): function

Used by the immune system to recognize foreign objects (antigens)

Antibody: variable region

Different in every antibody; contains the antigen binding site

Antibody: constant region

Same in every antibody of the same class

Epitope

The region of a protein that is recognized by an antibody

ELISA

Antibody-based method to detect/quantify a protein

Immunoprecipitation

Antibody-based method that pulls a specific protein out of a mixture

Proteomics

Large-scale study of the proteome (all proteins expressed in a cell)

Proteome

The spectrum of all proteins expressed in a cell

Proteomics: tool used

Mass spectrometry

Mass spectrometry: what does it give?

Size and quantity of ALL proteins in a cell extract

Proteomics: advantages

Quantifies all proteins in a cell; can also quantify other molecules and protein modifications

Proteomics: limitations

Cell localization info is lost; proteins must be cut (proteolyzed) and pieced back together

Proteomics vs Western blot: scale

Proteomics = large scale (all proteins); Western = small scale (specific protein)

Immunocytochemistry (ICC)

Uses an antibody + fluorophore to see where a protein is in fixed CELLS

Immunofluorescence / immunostaining

Other names for immunocytochemistry

ICC: step 1

Fix cells with a chemical that cross-links proteins and kills the cell

ICC: step 2

Permeabilize membranes with Triton X-100 (makes holes)

ICC: step 3

Incubate with antibody; it enters through holes and binds its epitope

ICC: step 4

Fluorophore on the antibody shows where the protein is

Fixative

Chemical that cross-links (covalent bonds between amino acids) and kills cells

Permeabilization

Making holes in the plasma membrane with non-ionic detergents (Triton X-100, digitonin)

Which detergent also permeabilizes the nuclear envelope?

Triton X-100

Direct immunocytochemistry

Fluorophore is attached directly to the primary antibody

Indirect immunocytochemistry: advantage

Signal amplification (more fluorophores per antigen)

Fluorophore

Small compound (0.2-1 kDa) that absorbs light and re-emits it at a longer wavelength (lower energy)

FITC

Fluorescein isothiocyanate; first common green fluorophore

Immunohistochemistry (IHC)

Uses antibodies to see where a protein is in a TISSUE sample

IHC: sample prep

Tissue is cut into thin sections with a microtome (frozen sections: cryostat)

IHC: detection types

Colorimetric (brown stain) or fluorescent

"Cyto" meaning

Cyto = cell

"Histo" meaning

histo = tissue

Microtome

Instrument that cuts thin sections of a tissue sample

Human Protein Atlas

Website showing protein expression patterns in tissues

WB vs ICC vs IHC: sample type

WB = cell extract; ICC = fixed cells; IHC = fixed tissue

WB vs ICC vs IHC: which tells protein SIZE?

Only Western blot

WB vs ICC vs IHC: which tell LOCATION?

ICC and IHC (not WB)

Widefield fluorescence microscopy

Fluorophore excited by a specific wavelength; emitted light at longer wavelength collected through emission filter

Widefield fluorescence: other name

Epifluorescence microscopy

Resolution limit of light microscopy

About 200 nm

Resolution

Minimum distance at which 2 spots can be distinguished

Confocal microscopy: how it works

Laser + pinhole selects light from a single thin focal plane in a thick specimen

Confocal microscopy: advantage

Images thick tissue without cutting sections

Confocal: resolution

Still limited by light wavelength (~200 nm)

Super-resolution microscopy

Breaks the 200 nm barrier; resolves structures as small as ~10-20 nm

Super-resolution: what does it rely on?

Fast microscopes, optical tricks, and computational image reconstruction

Light microscope: best for

Basic cell and tissue structure; any tissue

TEM: best for

Cell ultrastructure; very thin samples

SEM: best for

Cell/tissue SURFACES; any sample

Fluorescence microscopy: requirement

Molecule must have a fluorescent tag

Magnification meaning

How big it looks

Resolution meaning

How close two points can be and still be distinguished

Studying DNA: point mutation in a known region

PCR + Sanger sequencing

Studying DNA: large chromosomal changes

Karyotype

Studying DNA: location of a DNA sequence

DNA FISH

Studying DNA: all mutations in an individual

Whole genome sequencing

PCR + Sanger sequencing: best for

Point mutations

Southern blot / PCR: best for

Large mutations like insertions/deletions

Karyotyping: best for

Large chromosomal translocations

DNA FISH

Fluorescent In Situ Hybridization; shows where a DNA sequence is located

Whole genome sequencing: requirements

DNA sample, sequencer, and lots of money

PCR and FISH: what do both need?

Sample DNA and a known sequence (for primers/probes)

Probe

Small nucleotide chain with sequence similarity to the DNA/RNA you want to visualize

Primer

Short nucleotide sequence that starts DNA synthesis (like the RNA primer in replication)

Studying RNA: one specific RNA

RT-PCR (or Northern blot / qPCR)

Studying RNA: location of RNA

In situ hybridization (ISH)

Studying RNA: all RNA in a sample

Bulk RNA sequencing

Studying RNA: all RNA in every cell

Single cell RNA sequencing

RNA ISH

Shows where an RNA is expressed in cells/tissues (e.g. fgf8a in zebrafish embryos)

RT-PCR / qPCR: limitation

Measures one RNA at a time

Bulk RNA-seq

Sequences RNA from the whole sample mixed together

Single cell RNA-seq: advantage

Reveals gene expression changes bulk RNA-seq would miss

Single cell RNA-seq: downside

Huge amounts of data (terabytes)

scRNA-seq step 1

Collect sample and dissociate into single cells

scRNA-seq step 2

Capture each cell in its own oil bubble

scRNA-seq step 3

Isolate RNA in each bubble and add a cell barcode

scRNA-seq step 4

Sequence all the RNA together

Tissue dissociation: mechanical methods

Mortar and pestle, scissors, syringe

Tissue dissociation: enzymatic methods

Papain, collagenase, trypsin, DNase, dispase

UMAP

Plot used in scRNA-seq to identify cell types

Model: In vivo

In a living organism; costs animal lives; last choice

Model: In vitro

Molecular assays (e.g. DNA-protein, protein-protein interactions)

Model: In cell culture

Exogenous DNA and protein added to cells

Model: Ex vivo

Benefits of in vivo without causing pain to the animal

Model: In silico

Computer simulation of biology

Endogenous

Originating from within an organism

Exogenous

Originating from outside an organism