Cell Biology Lab: EGFR Signaling, Protein Analysis, and Troubleshooting

Vocabulary practice flashcards covering cell culture, membrane protein classes, EGFR signal transduction, endocytic sorting, protein quantification, SDS-PAGE/Western blot troubleshooting, protein trafficking, and immunofluorescence.

A431 cells

A model cell line derived from an older woman that overexpresses EGFR and is deficient in the tumor suppressor p53; cultured in DMEM with $10\%$ fetal bovine serum and $1\%$ penicillin-streptomycin at $37\,^\circ\text{C}$ with $5\%\,\text{CO}_2$.

Protein domains

Compact, independently folding units within a polypeptide chain that perform a specific functional role.

Integral membrane proteins

Proteins embedded directly into the lipid bilayer, exemplified by receptors like EGFR.

Peripheral membrane proteins

Proteins attached non-covalently to the membrane surface via interactions with lipids or integral membrane proteins, such as clathrin adaptors.

Lipid-anchored proteins

Proteins covalently attached to a lipid embedded within a single membrane leaflet, exemplified by RAS.

BCA Assay

A bicinchoninic acid protein quantification assay where peptide bonds reduce $\text{Cu}^{2+}$ to $\text{Cu}^+$ in alkaline conditions, forming a purple complex that absorbs at $562\,\text{nm}$.

Clathrin-mediated endocytosis

An endocytic pathway utilizing clathrin and AP2 machinery that operates across all EGF doses to recycle EGFR back to the cell surface.

Non-clathrin endocytosis

A clathrin-independent endocytic pathway utilizing Cbl and Grb2 machinery triggered at high EGF doses that targets EGFR for lysosomal degradation.

Receptor Tyrosine Kinases (RTKs)

A prominent subclass of enzyme-coupled receptors, including EGFR, whose intracellular kinase domains become active upon ligand-induced dimerization.

Trans-autophosphorylation

The reciprocal phosphorylation of tyrosine residues on the cytoplasmic tail of dimerized receptor kinase domains, generating docking sites for downstream signaling molecules.

MAPK pathway

A downstream proliferation cascade initiated when an RTK activates Ras through the adaptor protein Grb2.

PI3K/Akt pathway

A downstream cell growth cascade activated when RTKs stimulate PI3-kinase to phosphorylate inositol phospholipids, generating docking sites for Akt.

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; a method that separates proteins primarily by molecular weight to isolate a target protein from cellular lysates.

Coomassie gel staining

A staining technique that provides a visual readout of the total protein profile across samples, functioning as a critical loading control.

Western blotting

An analytical technique that uses primary antibodies to selectively detect the presence and relative abundance of specific target proteins (such as EGFR, Akt, and MAPK).

"Smiling" bands

A Western blot or SDS-PAGE artifact characterized by curved bands, typically caused by running the gel too fast or at an excessively high temperature.

Smeared lanes

A Western blot artifact caused by overloaded protein samples, degraded lysates, or incompletely denatured proteins.

White spots or patches (Western blot)

A membrane transfer issue resulting from air bubbles trapped between the gel and the transfer membrane during electroblotting.

Importins

Nuclear import receptors that recognize and bind nuclear localization signal (NLS)-bearing proteins to direct cargo through nuclear pores.

COPI and COPII

Vesicle coat proteins responsible for intracellular trafficking; COPI facilitates retrograde transport from the Golgi to the ER, while COPII mediates anterograde transport from the ER to the Golgi.

Indirect immunofluorescence

A detection method using an unlabeled primary antibody followed by a fluorophore-conjugated secondary antibody, offering higher sensitivity via signal amplification.

DAPI

A counterstain that binds DNA and emits blue fluorescence under UV light, used to define nuclear shape and cellular boundaries.

Photobleaching

The photochemical destruction and fading of fluorophores during imaging, counteracted by adding antifade reagents and restricting light exposure.

Co-localization

The overlapping signal of two fluorescent channels indicating spatial proximity and shared localization of proteins (such as EGFR with endosomal EEA1 or lysosomal LAMP1).