cell bio fall

In thermodynamics, what condition of free energy change indicates that a cellular process occurs spontaneously?

G<0

Why are polar molecules considered hydrophilic in biological systems?

They readily form hydrogen bonds and electrostatic interactions with water molecules, yielding a favorable G<0

Why do nonpolar molecules aggregate together in an aqueous cellular environment?

Interacting with each other rather than water minimizes energetically unfavorable interactions, resulting in a favorable G<0

How do histones overcome electrostatic repulsion to successfully package genomic DNA?

Positively charged histone surfaces form favorable electrostatic interactions with negatively charged DNA phosphate backbones

Why can hydrophobic hormones easily diffuse across plasma membranes while small inorganic ions cannot?

Membranes possess hydrophobic interior cores that make ion passage energetically unfavorable without transport proteins

How does a eukaryotic cell primarily generate and maintain compartmental specificity?

By selectively targeting specific functional proteins to distinct membrane-bound intracellular compartments

What is the main biological function of the cytoskeleton protein network in eukaryotic cells?

It provides structural framework regulating intracellular spatial organization, cell shape, and movement

What is the technical definition of a whole cell lysate?

A fluid containing total cellular contents produced by breaking all internal and external cellular membranes using harsh conditions

How is a subcellular lysate prepared without disrupting internal organelles?

Gentle cell lysis conditions break only the outer plasma membrane while leaving organellar membranes intact for differential fractionation

What general centrifugal physical principle governs differential subcellular fractionation

Smaller subcellular structures require higher centrifugation forces and speeds to sediment into a pellet

Which subcellular components pellet out under low-speed centrifugation during cell fractionation?

Whole unlysed cells, nuclei, and cytoskeletal structures form the initial low-speed pellet

Which organelles are sedimented together into a pellet under medium-speed centrifugation

Mitochondria, lysosomes, and peroxisomes pellet together during medium-speed centrifugation

What cellular components are pelleted out during very-high speed centrifugation

Ribosomes, viruses, and large macromolecular protein complexes sediment at very high speeds

What structural feature gives SDS its amphipathic detergent properties?

It contains a long hydrophobic hydrocarbon tail and a hydrophilic negatively charged sulfate head group

What is the specific role of SDS during protein sample preparation for gel electrophoresis?

It denatures non-covalent protein interactions and coats polypeptides uniformly with negative charges

What specific chemical action does B-mercaptoethanol perform on protein samples?

It reduces covalent disulfide linkages [S-S bonds] within or between polypeptide chains

Approximately how much SDS binds per mass unit of target protein during sample heating?

Approximately 1.4 grams of SDS binds per 1.0 gram of protein

Why do all SDS-coated polypeptides migrate at nearly identical speeds in free aqueous solution lacking a gel matrix?

SDS treatment equalizes both the charge-to-mass ratio and extended rod-like shape across all polypeptides.

How does a polyacrylamide gel matrix enable size-based separation of proteins during SDS-PAGE?

The molecular pore network exerts a sieving drag that retards larger polypeptides more than smaller ones

Directionally, toward which electrode do SDS-coated polypeptides migrate during electrophoresis?

Because SDS imparts a uniform negative charge, polypeptides migrate toward the positively charged anode

What chemical method is used to visualize all total proteins directly within an SDS-PAGE gel?

Staining the polyacrylamide gel with a general non-specific protein dye

Antigen

Any specific molecule or protein that is targeted and recognized by an antibody

Epitope

The precise molecular region or sequence on an antigen to which an antibody specifically binds

How does a monoclonal antibody differ in specificity from a polyclonal antibody?

A monoclonal antibody targets a single specific epitope, whereas a polyclonal antibody binds multiple distinct epitopes on an antigen

Why are most linear protein epitopes composed of a continuous stretch of amino acids?

Desaturation treatments like SDS unfold native tertiary structures into continuous linear amino acid sequences

What step must occur immediately after SDS-PAGE separation to perform western blotting?

Separated proteins must be transferred from the polyacrylamide gel onto a solid membrane support

What is the direct target bound by the primary antibody in a western blot assay?

The primary antibody specifically binds to the immobilized target antigen protein on the membrane

What is the direct target bound by the secondary antibody in a western blot assay?

The secondary antibody specifically binds to the constant region of the primary antibody

Why are polyclonal secondary antibodies preferred over monoclonal ones in Western blot detection?

Multiple polyclonal secondary antibodies can bind one primary antibody, causing substantial signal amplification

How does an enzyme-conjugated secondary antibody generate a detectable signal on a western blot membrane?

It catalyzes a chemical reaction converting an added substrate into a detectable colorimetric or chemiluminescent output

Flurophore

A specific functional group or protein domain responsible for absorbing and re-emitting light at characteristic wavelengths

How does the excitation wavelength of a fluorescent molecule compare to its emitted wavelength

The emission wavelength is always longer than the excitation wavelength due to vibrational energy loss

What cellular structure is selectively stained using the fluorescent dye DAPI?

DAPI selectively binds to genomic DNA located inside the nucleus

What is the purpose of chemical fixation in immunofluorsescence microscopy?

It cross-links and immobilizes cellular proteins to lock them into their physiological spatial locations

Why is membrane permeabilization necessary prior to staining intracellular proteins with antibodies?

Large antibody molecules cannot penetrate intact cell membranes without created membrane pores

What is the required chronological order of chemical processing steps for intracellular immunofluorescence?

Cells must be fixed first to preserve structure before being permeabilized for antibody entry

What major physiological limitation is associated with immunofluorescence microscopy

Fixation kills the cell, preventing the direct real-time study of dynamic intracellular processes

What is the molecular weight and original biological source of Green Fluorescent Protein GFP?

GFP is a 27 kDa protein originally discovered in the Aequorea jellyfish

What key advantage does GFP tagging offer over standard indirect immunofluorescence?

GFP allows real-time visualization of protein dynamics in living cells without fixation or exogenous antibodies.

How is a GFP fusion protein constructed inside a host cell?

Through genetic engineering that links the coding sequence of GFP in-frame with the target gene

Where on a target protein can a GFP tag be genetically fused

GFP can be fused to either the N-Terminus or C-Terminus of the target polypeptide

What potential scientific artificial can occur when expressing a GFP fusion protein?

The appended GFP tag may alter the native tertiary folding, activity, or localization of the protein

How do neutrophils navigate toward invading bacterial targets during innate immune responses?

Plasma membrane receptors. Detect bacterial chemotactic signals and trigger local cytoskeletal reorganization

How are internalized bacteria degraded within neutrophils following phagocytosis?

Phagocytosed vesicles are trafficked into fusion with degradation lysosomal compartments via vesicular transport

How does polyacrylamide gel pore size change when increasing the cross-linking density?

Higher cross-linking density decreases pore size, slowing down the migration of smaller protein molecules

What parameter is primarily analyzed when observing protein bands on a standard SDS-PAGE gel?

The relative molecular weight or mass of separated polypeptide chains

What unique spatial info does fluorescence microscopy provide that western blotting cannot?

It reveals the precise subcellular localization and distribution of target proteins within intact cells

What limitation prevents SDS-PAGE and Western blotting from assessing live cell dynamics?

Both procedures require complete cell lysis and protein denaturation prior to analysis

Why is a secondary antibody labeled with a fluorophore useful in multi-color fluorescence imaging?

Distinct fluorophores emit unique wavelengths, allowing simultaneous visualization of multiple distinct proteins

Why does the myelin membrane contains a lower protein content compared to typical plasma membranes?

To provide an effective electrical insulating barrier with a high proportion of lipids

Why does the inner mitochondrial membrane have a particularly high protein content?

To accommodate the high concentration of protein complexes involved in electron transport and ATP synthesis

What property defines an amphipathic molecule?

Possessing both polar [hydrophilic] and nonpolar [hydrophobic] regions within the same structure

Why is the interaction btwn water and a hydrophilic molecule energetically favorable

Hydrophilic molecules form electrostatic interactions and hydrogen bonds with surrounding water molecules

Why is dissolving a nonpolar molecule in water energetically unfavorable?

Water cannot form hydrogen bonds with nonpolar molecules, forcing water into highly ordered cage-like structures around them

What lipid shape favors the assembly of a spherical monolayer micelle in water?

Cone-shaped lipids containing a single hydrophobic tail

Why do two-tailed phospholipids spontaneously form lipid belayers instead of micelles?

Their cylindrical overall shape prevents packing into a tight sphere, favoring planar sheet alignment

Why is a flat, open planar lipid bilayer energetically unfavorable in an aqueous solution?

The hydrophobic fatty acid tails along the planar edges are directly exposed to water

How does a planar lipid bilayer eliminate its exposed hydrophobic edges to reach an energetically favorable state?

It spontaneously bends and seals itself into a closed spherical compartment [lipsome or vesicle]

What are three major classes of membrane lipids in animal cells?

Phospholipids, glycolipids, and sterols

What key chemical feature defines all phospholipids?

A hydrophilic head containing a phosphate group linked to a glycerol or sphingosine backbone

What backbones structure forms the basis of glycolipids?

Sphingosine

On which leaflet of the plasma membrane are glycolipids exclusively located?

The non-cytosolic [extracellular] leaflet

What is the primary form of sterol present in animal cell membranes?

Cholesterol

What are the three distinct structural regions of a cholesterol molecule?

A polar hydroxyl [-OH] head group, a rigid planar steroid ring structure, and a single short nonpolar hydrocarbon tail

How does a cis double bond affect the shape of an unsaturated fatty acid tail?

It introduces a rigid kink into the hydrocarbon backbone

How do straight, saturated fatty acids tails impact membrane lipid packing?

They pack closely together, making the membrane thicker and less fluid

How do kinked, unsaturated fatty acid tails increase membrane fluidity?

The kinks take up more space and prevent tight packing, weakening inter-tail van der Waals interactions

What double-bond configuration is predominantly found in biological unsaturated fatty acids?

The cis configuration

Name three rapid lipid movements that occur frequently within a single membrane leaflet

Lateral diffusion, flexion, and rotation

What is the term for the movement of a lipid molecule from one membrane leaflet to the opposite leaflet?

Flip-flop [or transverse diffusion]

Why does spontaneous lipid flip-flop between leaflets rarely occur?

Moving a hydrophilic head group through the central hydrophobic core requires a very high thermodynamic energy barrier

Which major phospholipids are predominantly localized to the cytosolic leaflet of the plasma membrane?

Phosphatidylethanolamine PE and phosphatidylserine PS

Which phospholipid concentrated in the cytosolic leaflet carries a net negative charge?

Phosphatidylserine PS

Which lipid types preferentially aggregate due to mutual high affinity to form microdomains known as lipid rafts?

Glycolipids and cholesterol

Why are lipid rafts thicker than surrounding membrane regions?

Glycolipids usually possess longer and predominantly saturated fatty acid tails

How do lipid rafts selectively enrich specific transmembrane signaling proteins?

Proteins with longer transmembrane domains preferentially partition into the thicker lipid raft domains

Why is the lipid bilayer impermeable to inorganic ions and hydrophilic molecules?

The central hydrophobic core creates an energetically unfavorable environment for charged or polar substances to cross

How does the resting cytosolic concentration of free calcium compare to its concentration in the extracellular space or ER lumen?

Cytosolic calcium is maintained at approximately ten thousand times lower concentration

What membrane lipid acts as the precursor for steroid hormones?

Cholestrol

What phospholipid serves as the biosynthetic precursor for endocannabinoids?

Phosphatidylethanolamine PE

What phospholipid acts as the parent precursor for all phosphoinositides PtdIns?

Phosphatidylinositol PI

On which membrane leaflet are phosphoinositides almost exclusively located?

The cytosolic leaflet

At which carbon positions on the inositol ring of phosphatidylinositol can phosphate groups be reversible added?

Positions three, four, and five

How do distinct phosphoinositide species recruit specific intracellular proteins to targeted membranes

Proteins containing specific phosphoinositide-binding domains selectively bind to membranes enriched in matching PtdIns species

What phosphoinositide substrate is specifically cleaved by activated Phospholipase C [PLC]?

Phosphatidylinositol 4,5-biphosphate (PI(4,5)P2)

What two signaling products are generated when Phospholipase C cleaves PI(4,5)P2?

Diacylglycerol [DAG] and Inositol 1,4,5-triphosphate [IP3]

What is the difference in cellular localization between DAG and IP3 following cleavage by PLC?

DAG remains anchored in the plasma membrane, whereas IP3 diffuses freely through the cytosol

What defines an integral membrane protein?

A protein with part of its polypeptide sequence directly embedded within or spanning the hydrophobic core of the lipid bilayer

What protein secondary structure most commonly spans the hydrophobic core in single-pass transmembrane proteins?

A alpha helix

Approximately how many hydrophobic amino acid residues are required for an alpha helix to span a typical five nm lipid bilayer

Twenty to thirty amino acids

Why are proline and charged amino acid residues extremely rare inside single-pass transmembrane alpha helices?

Proline introduces rigid kinks that disrupt alpha helical hydrogen bonding, and charged side chains are energetically unfavorable in the hydrophobic core

What defines a lipid-anchored membrane protein?

A protein associated with the membrane via a covalently linked lipid molecule embedded in the hydrophobic bilayer.

To which terminus of a protein is a GPI anchor covalently attached, and on which leaflet is it located?

Attached to the c-terminus, located exclusively on the non-cytosolic [extracellular/luminal] leaflet

What defines a peripheral membrane protein?

A protein bound to the membrane through non-covalent interactions with membrane lipids or integral proteins, without entering the hydrophobic core

What covalent modification commonly stabilizes extracellular domains of transmembrane proteins but is largely absent in cytosolic domains?

Disulfide bonds between cysteine residues

Why are disulfide bonds rarely formed in cytosolic domains of membrane proteins?

The cytosol is a reducing environment that keeps cysteine sulfhydryl groups reduced

What is a protein domain?

A structurally independent unit of a polypeptide chain that can fold and function autonomously

What does a hydropathy plot analyze along a protein’s primary amino acid sequence?

The average hydrophobicity index of successive amino acid segments to predict transmembrane regions

How does a transmembrane a-helix typically appear on a hydropathy plot?

As a continuous peak of positive hydrophobicity values spanning twenty to thirty amino acids

Why can a hydropathy plot fail to predict transmembrane domains in multipass B-barrel proteins?

The amino acid side chains in B-strands alternate between hydrophobic and hydrophilic, preventing extended fully hydrophobic peaks