MCB EOT EXAM

Light Microscopy

Uses light to visualize live or fixed specimens.

Bright Field Microscopy

Observes live, unstained cells using light phase shifts.

Chromogenic Stains

Dyes used to enhance contrast in fixed specimens.

H & E Staining

Haematoxylin stains DNA blue/purple; eosin stains proteins pink.

Fluorescence Microscopy

Uses fluorescent dyes to visualize specific cell components.

MitoTracker

Fluorescent dye for labeling mitochondria in cells.

LysoTracker

Fluorescent dye for labeling lysosomes in cells.

Colocalisation

Presence of two fluorescent signals in the same area.

Immunofluorescence Microscopy

Combines antibodies with fluorescent tags for specific detection.

Direct Immunofluorescence

Fluorescently labeled primary antibody binds directly to target.

Indirect Immunofluorescence

Uses secondary antibodies for signal amplification.

Epitope

Antigenic determinant recognized by an antibody.

Myc Epitope

Specific epitope used for tagging proteins in studies.

Phase Contrast Microscopy

Enhances contrast using refracted and un-refracted light.

Differential Interference Contrast

Uses two light beams for enhanced contrast imaging.

Microtome

Instrument for slicing specimens into thin sections.

Paraformaldehyde

Common fixative used for preserving cellular structures.

Sensitivity in Immunofluorescence

Ability to detect low levels of target proteins.

Cost of Antibodies

Conjugated primary antibodies are more expensive than secondary.

Signal Amplification

Multiple secondary antibodies bind to enhance detection.

Cell Sorting Methods

Techniques for separating cells based on specific criteria.

Myc

A proto-oncogene acting as a transcription factor.

Epitope Tag

A peptide sequence added for antibody detection.

Recombinant DNA Technology

Method for introducing specific DNA sequences into organisms.

Permeabilization

Process to allow antibodies access to cells.

Fluorescence Microscope

Instrument for visualizing fluorescently tagged proteins.

Peroxisomes

Organelles involved in lipid metabolism and detoxification.

Cytoplasm

Fluid inside cells excluding organelles and nucleus.

PEX13

Peroxisomal membrane protein with cytoplasmic C-terminus.

Transfection

Introducing foreign DNA into eukaryotic cells.

Lipofection

DNA delivery method using lipid vesicles.

Electroporation

Technique using electric pulses to facilitate DNA entry.

Triton X-100

Detergent that permeabilizes all cellular membranes.

Digitonin

Detergent that selectively permeabilizes cholesterol-rich membranes.

Confocal Microscopy

Technique for enhanced imaging using focused laser light.

Transmission Electron Microscope (TEM)

Microscope for observing internal structures at high resolution.

Diaminobenzidine (DAB)

Substrate oxidized to visualize peroxisomes in microscopy.

Scanning Electron Microscope

Microscope for viewing surfaces of unsectioned specimens.

C-terminus

End of a protein with a free carboxyl group.

Localization

Determining the specific location of proteins in cells.

Antibody Access

Ability of antibodies to bind to target proteins.

Green Dots

Indicate localization of PEX13-myc in microscopy.

Confocal Aperture

Pinhole that allows light from a specific plane.

Electron Density

Variation in electron scattering used in electron microscopy.

Protein Sorting Pathways

Two main routes for directing proteins to destinations.

Protein Sorting

Directs proteins to correct cellular compartments.

Targeting Signal

Short peptide guiding proteins to locations.

Specific Receptor

Recognizes and binds targeting signals.

Translocon

Channel for proteins to traverse membranes.

Cytosolic Ribosomes

Site of protein translation in cytoplasm.

Co-translational Translocation

Translation and translocation occur simultaneously.

Post-translational Translocation

Translation completes before translocation occurs.

Nuclear Pore Complex (NPC)

Large structure facilitating nuclear protein transport.

FG Nucleoporins

Proteins forming a gel-like matrix in NPC.

Nuclear Localization Signals (NLS)

Signals directing proteins to the nucleus.

GTPase Cycle

Molecular switch regulating protein interactions.

Importin Pathway

Mechanism for nuclear import of proteins.

Mitochondrial Targeting Sequences (MTS)

Signals directing proteins to mitochondria.

Translocase of Outer Membrane (TOM)

Complex facilitating mitochondrial outer membrane translocation.

Translocase of Inner Membrane (TIM)

Complex facilitating mitochondrial inner membrane translocation.

Energy Requirement

Energy needed for protein translocation processes.

Chaperones

Assist in proper folding of precursor proteins.

Nuclear Transport Factor 2 (NTF2)

Recycles Ran protein back into the nucleus.

Ribosome Attachment

Ribosomes bind to ER for co-translational translocation.

Sorting Defects

Errors in protein sorting causing diseases.

Precursor Proteins

Proteins synthesized in cytoplasm for organelle import.

Membrane Bound Translation

Translation occurs on ribosomes attached to membranes.

Hydrophobic Gel-like Matrix

Regulates transport through the NPC channel.

TOM

Translocase of Outer Membrane for mitochondrial proteins.

TIM

Translocase of Inner Membrane for mitochondrial proteins.

Proton motive force

H^+ gradient driving mitochondrial protein import.

ATP

Energy source for chaperone function in import.

MTS

Matrix Targeting Sequence for mitochondrial proteins.

Sub-mitochondrial compartments

Different mitochondrial locations for protein targeting.

PTS1

Type 1 peroxisome targeting signal, tripeptide SKL.

Pex5

Cytosolic receptor for PTS1 signal.

PTS2

Type 2 peroxisome targeting signal near N-terminus.

Pex7

Receptor for PTS2 signal in peroxisomal import.

Pex19

Receptor for importing peroxisomal membrane proteins.

PMP70

Example of a peroxisomal membrane protein.

Catalase

Most abundant peroxisomal matrix protein, reduces H2O2.

Pex11

Protein required for peroxisome proliferation.

mPTS

Peroxisomal membrane targeting signal for Class I PMPs.

Pex3

Membrane anchor protein for cargo-loaded Pex19.

Zellweger syndrome

Disorder from mutations in PEX genes affecting peroxisomes.

Hyperoxaluria Type 1

Peroxisomal disorder due to protein mistargeting.

AGT

Alanine:glyoxylate aminotransferase, normally sorted to peroxisomes.

Ubiquitination

Process marking Pex5 for extraction from membrane.

Pex14 and Pex13

Docking complex for Pex5 at peroxisomal membrane.

Peroxisomal ghosts

Large, empty peroxisomes due to defective import.

AGT Loss

Causes calcium oxalate excretion and kidney stones.

Peroxisomal Import

Involves folded proteins and translocon.

Cytosolic Receptor

Importin facilitates protein import into peroxisomes.

Secretory Pathway

Involves ER, Golgi, lysosomes, and plasma membrane.

Signal Sequence

Directs protein to the endoplasmic reticulum.

Amino Acid Length

Signal sequences are 16-30 amino acids long.

Positive Amino Acids

Located near the N-terminus of signal sequences.

Hydrophobic Residues

Bind signal recognition particle during protein targeting.

Signal Peptidase

Cleaves signal peptide from precursor proteins.

Glycosylation

N-linked glycosylation is dolichol-mediated.