Handout 11: Proteome Analysis

Name the four levels of gene expression regulation identified in the text.

Transcriptional, post-transcriptional, translational, and post-translational regulation.

Why does mRNA profiling often fail to accurately predict actual protein levels?

Regulation occurring after transcription significantly alters protein abundance and activity.

What biological information can be captured by the proteome but not the transcriptome?

Post-translational modifications such as phosphorylation, glycosylation, and ubiquitination.

Mathematically, how is the total resolution of a 2D gel calculated?

Resolution of the first dimension (IEF) multiplied by the resolution of the second dimension (SDS-PAGE).

In 2D PAGE, what property is used to separate proteins in the first dimension?

The isoelectric point (pI).

In 2D PAGE, what property is used to separate proteins in the second dimension?

Molecular weight (size).

Isoelectric Point (pI)

The pH at which a protein carries a net charge of zero.

What is the net charge of a protein when the local pH is below its isoelectric point (pI)?

Net positive charge.

What is the net charge of a protein when the local pH is above its isoelectric point (pI)?

Net negative charge.

Why does a protein stop moving in an IEF gel once it reaches its pI?

The protein becomes neutral and no longer experiences a force from the electric field.

What determines a protein's specific isoelectric point (pI)?

Its amino acid sequence and the number/pKa of its acidic and basic side chains.

What are ampholytes?

Small zwitterionic molecules that arrange themselves to form a stable, continuous pH gradient.

How do Immobilized pH Gradient (IPG) strips differ from traditional tube gels?

Ampholytes are covalently attached to the polyacrylamide gel on a plastic backing.

State one advantage of using IPG strips over carrier-ampholyte tube gels.

IPG strips provide higher reproducibility by preventing cathodic drift.

Toward which electrode does a protein migrate if it is loaded into a region where pH < pI?

The cathode (negative electrode).

Toward which electrode does a protein migrate if it is loaded into a region where pH > pI?

The anode (positive electrode).

What physical structure acts as the "molecular sieve" in SDS-PAGE?

A polyacrylamide gel matrix composed of cross-linked acrylamide and bis-acrylamide.

How does increasing the percentage of acrylamide affect the gel's resolution?

It creates smaller pores, improving the resolution of small proteins while hindering large ones.

Why is native charge a problem for separating proteins by size alone?

Native charge varies wildly and is not proportional to the protein's mass.

How does SDS ensure that migration is based only on molecular weight?

It unfolds proteins and confers a uniform negative charge-to-mass ratio, eliminating shape and native charge effects.

Approximately how many SDS molecules bind to a protein during denaturation?

One SDS molecule for every two amino acids.

What is the function of 8 M urea in the rehydration buffer?

It acts as a chaotrope to denature proteins and keep them soluble by preventing aggregation.

What is the function of 2% CHAPS in the sample buffer?

It is a zwitterionic detergent that solubilizes hydrophobic and membrane proteins without affecting IEF.

What is the role of Dithiothreitol (DTT) in the 2D PAGE protocol?

It is a reducing agent that breaks and prevents the formation of disulfide bonds.

Why are Bio-Lyte 3/10 ampholytes added to the rehydration buffer?

They help maintain the pH gradient and prevent protein precipitation at the edges of the strip.

What is the purpose of Bromophenol blue in the gel electrophoresis process?

It serves as a visual tracking dye to monitor the progress of the run.

In Equilibration Buffer II, what is the specific role of iodoacetamide?

It alkylates free sulfhydryl groups on cysteines to permanently prevent disulfide bond reformation.

Which reagent acts as the "glue" to connect the IPG strip to the SDS-PAGE gel?

Overlay agarose.

What are the components of the Fixing solution used after the SDS-PAGE run?

40% methanol and 10% acetic acid.

Why must SDS be washed out of the gel during the fixing step?

To prevent interference with the subsequent protein staining process.

Which protein stain is used for its high sensitivity and compatibility with mass spectrometry?

Sypro Ruby.

Why is the sample pipette distributed as a continuous line during strip rehydration?

To ensure even protein loading and complete rehydration of the IPG strip.

What is the consequence of air bubbles during the IPG strip rehydration step?

Bubbles cause non-uniform rehydration and disrupted current flow during focusing.

What is the function of water-saturated paper wicks placed over the electrodes?

They act as salt bridges to ensure consistent current and prevent electrical arcing.

Why is an IPG strip placed gel-side-down into the sample tray?

To allow the gel matrix to directly absorb the protein sample through passive rehydration.

What is the purpose of the mineral oil overlay during IEF?

It prevents evaporation of water and the crystallization of urea during the long run.

Why must IEF be performed at a constant temperature of 20C?

Temperatures above 30C cause urea to decompose into isocyanate, leading to protein carbamylation.

What is the negative effect of protein carbamylation on a 2D gel?

It modifies lysine residues, which changes the protein's pI and ruins the resulting spot pattern.

Why is a 50 microA current limit set per strip during IEF?

To prevent Joule heating, which can melt the gel or cause protein streaking.

What is the purpose of the initial 250V linear step in the IEF voltage program?

It allows gentle protein entry and removes salts to prevent precipitation.

The total "focusing power" of an IEF run is measured in which unit?

Volt-hours (Vh).

Why are focused IPG strips frozen at -70C if not used immediately?

To stop protein diffusion and preserve the gel without forming damaging ice crystals.

What is the primary objective of Equilibration Buffer I?

To introduce SDS for the second dimension and re-reduce any reformed disulfide bonds with DTT.

Why is DTT removed during the transition to Equilibration Buffer II?

To prevent excess DTT from interfering with the alkylation reaction or causing background noise.

What happens to the transfer of proteins if air bubbles are trapped between the IPG strip and the SDS gel?

It creates gaps in the conductive bridge, leading to missing spots, smiling, or streaking.

At what voltage is the SDS-PAGE (second dimension) typically run in this protocol?

200V

Why is the run stopped when the bromophenol blue reaches the bottom of the gel?

The dye represents the leading front, indicating that small proteins have migrated through the full gel length.

Which amino acid residues does Sypro Ruby primarily bind to?

Basic residues, specifically arginine, lysine, and histidine.

Why must gels stained with Sypro Ruby be kept in the dark or covered with foil?

To protect the fluorescent fluorophores from photobleaching.

What is the purpose of destaining the gel in 20% ethanol?

To lower background fluorescence so that protein spots are more clearly visible.

Concept: Orthogonal Separation

Definition: Using two independent, non-overlapping physical properties (like pI and size) to maximize resolution.

What is the role of glycerol in the equilibration buffers?

It increases the density of the buffer to help the strip settle and stabilize the gel during handling.

Why is the IPG strip drained vertically for 10 seconds after the IEF run?

To remove excess mineral oil before the equilibration steps.

In the human analogy for 2D PAGE, what two factors are compared to pI and size?

Age and height.

What is the approximate protein concentration of the E. coli sample provided in the kit?

1.35 mg/ml.

How does the electrical current typically change during a constant-voltage SDS-PAGE run?

The current drops as the run progresses.

What is the result of using a "rapid ramp" in the final voltage step of IEF?

It maximizes focusing efficiency to produce razor-sharp protein bands.

Why is passive rehydration performed for 16 hours before applying high voltage?

To allow the strip to swell slowly and ensure proteins diffuse into the gel matrix gently.

What component of the IEF cell provides temperature control?

The cooling/heating plate in the PROTEAN IEF cell tray.

How does the conductivity of the SDS-PAGE buffer contribute to the run?

It maintains a stable pH and provides the necessary ions to carry the electrical current.

Which specific detergent is used to solubilize proteins for IEF because it is zwitterionic?

CHAPS.

Why is SDS inappropriate for use in the first dimension (IEF)?

SDS is anionic and would give all proteins a negative charge, overriding their natural isoelectric points.

What visual evidence indicates that proteins have been "fixed" in the gel?

The proteins precipitate within the gel matrix, preventing them from diffusing out.

How is a digital image of a Sypro Ruby stained gel typically captured?

Using a Gel Doc system with a UV or blue light transilluminator.

What is the ultimate purpose of storing the finished gel in distilled water?

To prevent the gel from drying or cracking and keep it ready for future spot excision.

Describe the relationship between protein length and mass after SDS treatment.

There is a linear relationship between the mass of the protein and its length as a random-coil rod.

Why are linear chains of acrylamide cross-linked with bis-acrylamide?

To form the actual pores of the molecular sieve through which proteins migrate.

In IEF, where is the acidic end of the pH gradient located relative to the electrodes?

At the anode (+ electrode).

In IEF, where is the basic end of the pH gradient located relative to the electrodes?

At the cathode (- electrode).

What is the consequence of "cathodic drift" in older tube gel systems?

The pH gradient becomes unstable and drifts toward the cathode over time, reducing reproducibility.

What does a "spot train" on a 2D gel usually indicate?

Partial protein oxidation or incomplete alkylation of cysteine residues.