DNA Microarrays, 2D Gel Electrophoresis, and BIS-102 Review

Lecture Overview

This lecture began with course announcements and then continued a review of BIS-102 material needed for BIS-103, covering affinity chromatography, three types of analytical gel electrophoresis (IEF, native PAGE, and SDS-PAGE), fatty acid structure and nomenclature, and two classes of lipids (acylglycerols and phospholipids). The lecture then introduced two techniques for examining overall cellular metabolism: DNA microarrays for analyzing mRNA expression profiles, and 2D IEF-SDS PAGE for visualizing the full proteome. Both techniques were presented in the context of comparing cancer cells to normal cells.

Key Concepts and Definitions

• Affinity chromatography: A purification technique using a column of polar beads with covalently attached ligands that selectively bind a target molecule through weak bonds.
• Ligand (column context): A molecule covalently linked to the column arm that the target protein recognizes; can be a substrate, product, inhibitor, coenzyme, or cofactor.
• PAGE: Polyacrylamide gel electrophoresis.
• IEF (isoelectric focusing): A gel technique that separates proteins by their isoelectric point (PI); a gentle method that preserves all levels of protein structure.
• PI (isoelectric point): The pH at which the net charge on a protein is zero.
• Native PAGE: A gel technique that separates proteins by both size and charge-to-mass ratio; all levels of protein structure remain intact.
• Stacking gel: The upper portion of a PAGE gel that compresses a large sample volume into a thin band before it enters the resolving gel.
• Resolving gel: The lower, main portion of a PAGE gel where proteins separate.
• SDS (sodium dodecyl sulfate): A 12-carbon man-made strong detergent with a full minus-one charge; denatures proteins and gives every protein the same charge-to-mass ratio.
• SDS-PAGE: A gel technique that separates denatured proteins by size only; only primary structure remains intact.
• Saturated fatty acid: A fatty acid with no double bonds.
• Unsaturated fatty acid: A fatty acid with one or more double bonds.
• Palmitic acid (16:0): A saturated fatty acid with 16 carbons and zero double bonds.
• Stearic acid (18:0): A saturated fatty acid with 18 carbons and zero double bonds.
• Oleic acid (18:1,9): An unsaturated fatty acid with 18 carbons and one cis double bond beginning at carbon 9.
• Linoleic acid (18:2,9,12): An unsaturated fatty acid with 18 carbons and two cis double bonds beginning at carbons 9 and 12.
• Alpha-linolenic acid (18:3,9,12,15): An unsaturated fatty acid with 18 carbons and three cis double bonds beginning at carbons 9, 12, and 15.
• Acylglycerol: A class of lipid with fatty acids attached to a glycerol backbone; triacylglycerols are energy deposits commonly called fat.
• L-glycerol: The form of glycerol used by most organisms; the hydroxyl on carbon 2 is at the left. Archaea use D-glycerol.
• Phospholipid: A class of lipid involved in membrane construction; has a glycerol backbone with fatty acids at positions 1 and 2 and a phosphate group at position 3.
• Phosphatidylcholine: A common phospholipid with choline attached via a phosphodiester at carbon 3 of glycerol.
• Phosphatidylserine: A common phospholipid with the amino acid serine attached via a phosphodiester at carbon 3 of glycerol.
• Quaternary amine: A nitrogen with four groups attached; always carries a positive charge regardless of pH.
• DNA microarray: A technique for examining all mRNAs a cell is producing, using competitive hybridization of fluorescently labeled cDNAs to a chip bearing single-stranded gene sequences.
• Poly-A tail: A stretch of adenine nucleotides at the 3' end of mRNA; distinguishes mRNA from tRNA and rRNA.
• cDNA (copy DNA): A DNA strand synthesized from an mRNA template using reverse transcriptase.
• Reverse transcriptase: An enzyme that uses an RNA template to synthesize a complementary DNA strand; requires a double-stranded starting point and deoxynucleoside triphosphates.
• A260: Absorbance at 260 nm wavelength; used to detect nucleic acids via their conjugated double bonds in nitrogenous bases.
• Ampholytes: Small organic molecules added to an IEF gel that create a pH gradient when current is applied.
• TEMED: An organic molecule that prolongs the life of free radicals during gel polymerization.
• Ammonium persulfate: A compound whose persulfate bond breaks in water to generate free radicals that initiate acrylamide polymerization.
• Mercaptoethanol: A reducing agent that breaks disulfide bonds; used in SDS treatment to prevent artificial cross-linking.
• Clustered micelles: The structures formed when SDS-coated denatured protein subunits repel each other due to their cloud of negative charge.
• Proteome: The complete set of proteins expressed by a cell.
• Bromophenol blue: A tracking dye used in PAGE that runs faster than any protein and does not interact with proteins; signals when to stop electrophoresis.

Chronological Lecture Notes

1. Course Business

• Homework 1 has been posted; it is the only assignment for the first midterm.
• The homework covers a lot of material; students should not be surprised by its scope close to the exam date.
• Accommodated exam services confirmed; exams can only be taken between 8 a.m. and 5 p.m. on exam day.

Exam emphasis: Homework 1 is the only assignment for the first midterm and covers the full scope of testable material.

2. Review: Affinity Chromatography

• Microscopic beads with pores; the overall bead must be polar.
• Pores must be large so all biomolecules can enter and exit; the bead is not performing molecular sieving or separating by size.
• A covalent arm (organic structure) is attached to the bead; recommended length is six carbons.
  • Too short (one or two carbons): the arm will not function properly.
  • Too long (12+ carbons): the arm acts as a hydrophobic coating on the bead.
• The arm lifts the ligand (L) away from the bead surface so that a three-dimensional enzyme can reach around with its active site and grab the ligand.
• The ligand is covalently linked to the arm and cannot come off; typically a small organic molecule.
• Possible ligands: a substrate, product, inhibitor, coenzyme, or cofactor (metal ion).
• For enzymes requiring two substrates (e.g., hexokinase uses glucose and ATP), only one substrate is placed on the column; providing both would allow the enzyme to catalyze the reaction and break everything down.
• The enzyme recognizes the ligand at the active site through weak bonds: ionic bonds, hydrogen bonds, van der Waals forces, and the hydrophobic effect.
• The enzyme binds dynamically (coming on and off the ligand), but the large number of ligands on each bead keeps the enzyme retained on the column.
• All other molecules do not bind and flow right through.
• This technique is relevant to DNA microarrays (discussed later) and to isolating molecules for metabolic studies.

3. Review: Analytical Gel Electrophoresis -- IEF Gels

• PAGE = polyacrylamide gel electrophoresis.
• IEF (isoelectric focusing) gel separates proteins by their PI.
• This is a gentle technique: all levels of protein structure (primary, secondary, tertiary, quaternary) remain intact.
• If 20 bands appear, there are at least 20 proteins; some bands may contain multiple proteins that happen to share the same PI.
• Can be run as a horizontal slab gel or inside a vertical glass tube.
• No tracking dye is needed because proteins move to their PI and stop; the experiment is done when the current drops close to zero.

4. Review: Analytical Gel Electrophoresis -- Native PAGE

• Native PAGE separates proteins by two parameters: size (small proteins migrate faster) and charge-to-mass ratio (Q/M).
• A protein with a higher charge-to-mass ratio moves faster, even if it is larger.
• Running a single native PAGE gel does not reveal whether a faster-moving protein is smaller or simply has a higher charge-to-mass ratio.
• "Native" means all levels of protein structure are intact; subunits remain associated (e.g., lactate dehydrogenase, described as a four-subunit enzyme from the last step in glycolysis, stays intact on IEF or native gels).
• Run as a vertical slab gel between two thin glass plates.
• Gel structure:
  • Resolving gel (bottom, larger portion): where proteins separate.
  • Stacking gel (top, with sample wells): compresses the sample into a thin band before entering the resolving gel.
  • Wells are formed by inserting a Teflon comb into the stacking gel before polymerization; removing the comb leaves indentations (approximately 15 microliters capacity each).
• Bromophenol blue dye is added to the sample: it does not interact with proteins and runs faster than any protein, serving as a tracker to indicate when to stop electrophoresis and prevent proteins from running off the gel.
• Why stacking matters: Without stacking, the sample volume is too large and diffusion (entropy) causes bands to spread and overlap, preventing resolution. The stacking gel condenses the sample into a thin band so that bands remain sharp in the resolving gel.

Student question: What makes the stacking gel stack?

• The stacking gel, the resolving gel, and the upper buffer each have different pH values.
• Stacking requires a lead ion and a trailing ion that set up a voltage gradient.
• Standard recipe: resolving gel at pH 8.8, stacking gel at pH 6.8.
• The trailing ion is slow at the stacking gel pH but speeds up when the pH changes, passing the proteins.
• The lecturer noted this mechanism takes about 20 minutes to fully explain and is covered in MCB 120L.
• IEF gels do not need a stacking gel because proteins focus to sharp bands at their PI as long as power is applied; diffusion (entropy) only sets in after the power is turned off.

5. Review: Analytical Gel Electrophoresis -- SDS-PAGE

• SDS = sodium dodecyl sulfate, a man-made strong detergent.
• Strong detergents have a full charge (minus one or plus one); mild detergents have only polar groups.
• SDS denatures the entire protein structure and gives every protein the same charge-to-mass ratio.
• In SDS-PAGE, proteins separate by size only; charge-to-mass ratio is no longer a factor.
• Only primary structure (linear amino acid sequence) remains intact; secondary, tertiary, and quaternary structure are all destroyed.
• Why these three gels matter:
  • Purify enzymes and proteins.
  • Determine protein size and identify whether subunits are present and how big they are.
  • Gels are easy to run (one to two days).
  • 2D IEF-SDS PAGE (introduced later) enables examination of all proteins in a cell.

  Reference figure · external source

6. Review: Fatty Acids

• Fatty acids are parts of lipids, not lipids themselves.
• Five fatty acids to know, in two classes:
  • Saturated (no double bonds):
    • Palmitic acid: 16:0 (16 carbons, zero double bonds).
    • Stearic acid: 18:0 (18 carbons, zero double bonds).
  • Unsaturated (one or more double bonds):
    • Oleic acid: 18:1(9) -- one cis double bond between carbons 9 and 10.
    • Linoleic acid: 18:2(9,12) -- two cis double bonds, beginning at carbons 9 and 12.
    • Alpha-linolenic acid: 18:3(9,12,15) -- three cis double bonds, beginning at carbons 9, 12, and 15.
• Nomenclature: The first number is total carbons; the number after the colon is the number of double bonds; numbers in parentheses indicate the carbon positions where double bonds begin.
• Carbon 1 is the carbon of the carboxyl group.
• All double bonds in these fatty acids are cis: the chain continues on the same side of the double bond, creating a kink. The enzymes that form these double bonds are stereospecific and always produce the cis configuration.
• Ionization: If the carboxyl group (COOH) is ionized to COO-, the name changes (e.g., palmitic acid becomes palmitate).
• Free fatty acids act as detergents (SDS is 12 carbons with a negative charge; palmitate is 16 carbons with a negative charge). Only sick or dying cells have lots of free fatty acids; normally, fatty acids are covalently attached to other compounds to prevent detergent activity.
• Energy content: Stearic acid (18 carbons) contains far more energy per molecule than glucose (6 carbons); burning fatty acids requires proportionally more exercise per molecule.
• Cis double bond kinks affect membrane fluidity: Membranes with more kinked fatty acids can move more easily.
• BIS-103 will cover fatty acid breakdown (beta-oxidation) and possibly fatty acid synthesis.

Exam emphasis: On exams, students are typically asked to number the carbons in fatty acid structures.

7. Review: Lipids -- Acylglycerols

• Scientists classify lipids into seven classes; two are covered in BIS-103.
• Acylglycerols:
  • Glycerol backbone with fatty acids attached at carbons 1, 2, and 3 via ester bonds.
  • A triacylglycerol has three fatty acids (any of the five learned); commonly called "fat."
  • Triacylglycerols are not in membranes; they are energy deposits.
  • One triacylglycerol molecule contains a large amount of energy (three long fatty acids plus glycerol).
  • The body breaks down triacylglycerols for energy when food is not available (to feed the brain, muscles, etc.).
• L-glycerol: The hydroxyl on carbon 2 is written to the left. Most organisms use L-glycerol. Archaea use D-glycerol (reason unknown).
• Enzyme stereospecificity: A unique enzyme attaches a fatty acid to each position on glycerol (one enzyme for position 1, a different enzyme for position 2, a third for position 3). Different enzymes remove fatty acids from each specific position. Enzymes are very specific and very fast, so cells do not need many copies.
• Enzymes easily distinguish left-handed from right-handed molecules in three dimensions using multiple weak bonds (perhaps 20).

8. Review: Lipids -- Phospholipids

• Phospholipids are involved in making membranes (plasma membrane and organelle membranes such as Golgi apparatus).
• Structure: glycerol backbone with a fatty acid at carbon 1, a fatty acid at carbon 2, and a phosphate group at carbon 3; the phosphate is linked to an alcohol, forming a phosphodiester.
• Phosphodiesters are very common in cells (DNA and RNA backbones are sugar-phosphate-sugar-phosphate with phosphodiesters).
• Two phospholipids to know:
  • Phosphatidylcholine: Choline is attached via the phosphodiester. The nitrogen in choline has four groups attached (quaternary amine) and always carries a positive charge regardless of pH.
  • Phosphatidylserine: The amino acid serine is attached via the phosphodiester. Amino acids serve functions beyond building proteins.

Student question: Can phosphate only go on carbon 3?

• Yes. The enzyme that places the phosphate is stereospecific and never makes a mistake. If a mutation caused an error, it would likely be lethal and the progeny would not survive.

Exam emphasis: Students may be asked to draw specific phospholipids at a given pH with designated fatty acids at each position (e.g., "draw phosphatidylserine at pH 7 with palmitate at position 1 and stearic acid at position 2").

Reference figure · external source

9. DNA Microarrays -- Overview and mRNA Isolation

• DNA microarrays examine all the messenger RNAs that cells are making.
• Application example: comparing cancer cells to normal cells to identify what is going wrong.
• RNA is extracted from cells using an organic solvent.
• Only mRNA is desired; cells also contain tRNA, ribosomal RNA, and other types.
• mRNA is distinguished by its poly-A tail at the 3' end; tRNA and rRNA lack a poly-A tail.
• Nucleic acids are written 5' to 3'.
• An affinity column separates mRNA from other RNA types:
  • The ligand is polydeoxythymidine (oligo-dT), covalently attached directly to the bead (no arm is needed because mRNA is linear, not a three-dimensional molecule requiring active-site access).
  • mRNA poly-A tails bind to the oligo-dT through weak bonds (hydrogen bonding between A and T).
  • The binding is dynamic: bonds can break and reform, but the abundance of oligo-dT on the beads keeps the mRNA retained.
  • tRNA and rRNA flow through without binding.
• Two identical columns are run: one for cancer cell RNA, one for normal cell RNA (the control).
• The column apparatus includes a plastic fritted disc at the bottom with small holes that allow buffer and dissolved molecules through but trap the beads.

10. Column Measurement and Elution

• Nucleic acids absorb UV light due to conjugated double bonds in nitrogenous bases (e.g., adenine).
• Absorbance is measured at 260 nm (A260) using a spectrophotometer.
• Flow-through: The initial high-absorbance fractions contain tRNA and rRNA passing through; these are discarded.
• The column is washed with buffer at a pH that maximizes mRNA-ligand binding (determined by trial and error; approximately pH 7.5).
• Absorbance drops to zero as non-mRNA species are fully washed out.
• Elution methods to release mRNA from the column:
  • Change the pH by a large amount (disrupts hydrogen bonding holding the mRNA to the ligand).
  • Decrease the salt concentration.
• The eluted peak contains the mRNA population.

11. Break

• Five-minute break; the lecturer noted the next topic would be why lowering salt concentration causes mRNA to elute.

12. Why Decreasing Salt Concentration Elutes mRNA

• In any nucleic acid duplex, the sugar-phosphate backbone carries negative charges (PO-) along both strands at experimental pH values.
• These negative charges repel each other.
• Cations in solution (e.g., Mg2+, Zn2+, or positively charged protein residues like arginine and lysine) neutralize this repulsion and stabilize the duplex.
• Pouring through a buffer with fewer cations removes the charge shielding; the strands repel each other and separate.

13. cDNA Synthesis and Fluorescent Labeling

• The goal is to convert colorless mRNA into fluorescently labeled, stable DNA copies (cDNA).
• Procedure (for each mRNA population):
  1. Add oligo-dT primers in excess; these anneal to the poly-A tail to provide a double-stranded starting point (polymerases require a double-stranded region to begin).
  2. Add reverse transcriptase (purified enzyme, expensive).
  3. Add deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) as energy sources and building blocks for the DNA strand. Without them, no polymer can be made.
  4. One of the deoxynucleoside triphosphates (dATP, chosen arbitrarily) is made fluorescent: a chemical group is added that causes it to emit colored light at a longer wavelength when excited by light.
  5. Reverse transcriptase uses the mRNA as a template to synthesize a complementary DNA strand; every time a dATP is incorporated opposite a uridine in the template, some will carry the fluorescent label (not all need to be labeled).
• Color assignment:
  • Cancer cell mRNA --> red fluorescent cDNA.
  • Normal cell mRNA --> green fluorescent cDNA.
• Two important outcomes of this step: (1) the mRNA gains a visible color via its cDNA copy, and (2) the copy is made of DNA, which is much more stable than RNA.

Student question: Why dATP specifically rather than dGTP?

• The choice is arbitrary; any deoxynucleoside triphosphate could be made fluorescent. Not all copies are made fluorescent because it is too expensive.

14. Removing the mRNA Template

• The cDNA must be made single-stranded to hybridize with the microarray chip, so the mRNA template must be destroyed.
• Method: Incubate the mRNA-cDNA duplex in 1 M NaOH (strong base, very high concentration) with heat for 15 minutes.
• Mechanism of RNA degradation:
  • OH- removes a proton from the 2'-hydroxyl of the RNA sugar, generating a 2'-O-.
  • The nearby phosphorus atom has a partial positive charge because all surrounding oxygens are electronegative and pull electron density away from it.
  • The 2'-O- attacks the phosphorus in a substitution reaction, breaking the phosphodiester bond.
  • This process occurs at every nucleotide along the mRNA chain, completely fragmenting it.
• DNA is unaffected because it lacks a 2'-hydroxyl group (it has only a hydrogen at the 2' position).
• Critical neutralization step: After mRNA destruction, HCl is added to bring the pH back down to 7. At high pH, cDNA cannot form the hydrogen bonds needed to bind the microarray chip.

15. The DNA Microarray Chip

• A microscope slide with a chip containing tiny dots of single-stranded DNA; costs approximately $800 per slide.
• Each dot contains multiple identical copies of a single gene's DNA sequence, deposited in approximately one-nanoliter volumes by machines (too small to pipette manually).
• Adjacent dots represent different genes.
• Each dot must have approximately the same number of DNA copies for fairness.
• A single chip might represent one chromosome.
• The single-stranded DNAs act as receptors.
• Only the manufacturing company knows the identity of each gene at each dot position; the researcher is paying for that information.

Reference figure · external source

16. Hybridization, Scanning, and Interpretation

• Equal amounts of red cDNA (cancer) and green cDNA (normal) are pipetted into a container with the chip and buffer (optimized by the company for pH and conditions).
• The mixture is incubated with rotation on a platform; cDNAs compete for binding spots (compared to musical chairs).
• After incubation, the slide is rinsed to remove non-specific binding, then placed in a fluorescence scanner.
• The scanner quantifies color at each spot:
  • Red: More cancer-cell cDNA bound; gene is more expressed in cancer cells.
  • Green: More normal-cell cDNA bound; gene is more expressed in normal cells.
  • Yellow: Approximately equal red and green; gene is expressed equally in both cell types.
  • Black: No cDNA bound; gene is not expressed in either cell type.
• The scanner can image red and green channels separately and merge the two images.
• Yeast example (from Lehninger): Green spots represented genes expressed during normal growth; red spots represented genes expressed during spore formation; yellow spots indicated genes active in both conditions; many black spots indicated unexpressed genes.
• Significance for cancer research: Red spots identify genes whose expression differs in cancer cells; not all may directly cause cancer -- one upstream gene could control many downstream targets.
• DNA microarrays show all genes simultaneously, a major advantage over older approaches that isolated one thing at a time.
• One homework question asks students to explore this technique further.

17. Transition to 2D IEF-SDS PAGE -- Why Proteins Matter Too

• 2D IEF-SDS PAGE looks at all proteins (the proteome) rather than mRNAs.
• Advantage over microarrays: A single base substitution in DNA may not be detected by a microarray because the rest of the polymer still binds and the spot shows a color. However, if that substitution causes a charge change in the encoded protein, the IEF dimension of a 2D gel or a native PAGE gel will detect the shift.
• SDS-PAGE alone will not detect charge changes because SDS coats everything uniformly.

18. Making the IEF Tube Gel (First Dimension)

• A glass tube is stoppered at one end; the gel mixture is poured in and polymerizes in about 15 minutes into a clear gel.
• Components:
  • Acrylamide: Monomer for the gel matrix.
  • Bisacrylamide: Creates cross-links; without it, no gel forms (just a watery solution).
  • Ampholytes: Small organic molecules that create the pH gradient when current is applied; purchased in small, expensive bottles for specific pH ranges (e.g., pH 2 to 10 to cover all proteins).
  • TEMED: Prolongs the life of free radicals needed for polymerization; without it, free radicals decay before initiating the chain reaction and no gel forms.
  • Ammonium persulfate: The persulfate bond (O-O between two sulfate groups) is very unstable and breaks almost instantly in water, generating two sulfate free radicals, each with a single unpaired electron. These free radicals initiate acrylamide polymerization: a free radical attaches to a monomer, which can then attack another monomer, propagating a chain reaction that forms the gel network.

19. Running the IEF Tube Gel

• The apparatus uses two electrode solutions:
  • Top (negative electrode): 60 mM NaOH (high pH).
  • Bottom (positive electrode): 60 mM H2SO4 (low pH).
• These extreme pH values trap the ampholytes within the gel.
• For analyzing all proteins, a broad pH gradient is used (pH 2 to 10).
• The protein sample is loaded on top of the gel.
• When current is applied, proteins migrate to their isoelectric point and form sharp bands.
• A gel analyzing all proteins from a cell may show a hundred or more bands; some proteins may co-migrate at the same PI.
• This is the first dimension: separation by PI.

20. SDS Treatment of the Extruded IEF Gel

• The polymerized IEF gel is extruded from the glass tube using a small plunger (comes out like a thin piece of spaghetti) into a tray of SDS solution. The same is done for the normal-cell gel.
• SDS is a small molecule that can diffuse into the gel; proteins are too large to diffuse out.
• SDS structure: 12-carbon nonpolar tail (which can rotate freely in solution); charged head group with a minus-one charge.
• Strong detergent = full charge; distinguished from mild detergents that have only polar groups.
• SDS treatment (with mercaptoethanol and heat):
  • Separates subunits of multi-subunit proteins.
  • Completely unfolds each subunit (only primary structure remains; no alpha helices, beta-pleated sheets, tertiary structure, or biological activity).
  • Mercaptoethanol (added in excess) reduces disulfide bonds; this is necessary because atmospheric oxygen can create artificial disulfide bonds in the lab, potentially cross-linking subunits or parts of a single subunit.
  • Heat accelerates the denaturation.
• SDS binding: The hydrophobic tail of SDS binds to hydrophobic amino acid R groups (leucine, isoleucine, tryptophan, etc.); it does not bind to charged or polar residues (e.g., glutamate with a negative charge, arginine with a positive charge), leaving gaps.
• Charge-to-mass equalization: A larger subunit binds proportionally more SDS molecules. The thousands of negative charges from SDS overwhelm any native protein charge (e.g., a native charge of +8 becomes irrelevant). All proteins end up with the same charge-to-mass ratio.
• SDS-coated denatured subunits repel each other due to their clouds of negative charge, forming clustered micelles.

21. Running the Second Dimension (SDS-PAGE Slab Gel)

• The SDS-soaked IEF gel is placed on top of an SDS-PAGE vertical slab gel.
• Instead of a comb with multiple small wells, a single large indentation is created using a Teflon comb to accommodate the entire IEF gel.
• Gel construction: Pour the resolving gel first (it polymerizes), then pour the stacking gel on top. Place the IEF gel into the single large well.
• Gel composition: Stacking gel at approximately 4%; resolving gel at approximately 10%.
• SDS is present throughout the gel.
• Electrode placement: The positive electrode must be at the bottom because all SDS-coated proteins are heavily negative and must migrate downward toward the positive electrode.

Exam emphasis: Choosing the correct electrode placement for SDS-PAGE is a likely first midterm question. Reasoning: SDS-coated proteins are super negative and must migrate toward the positive electrode at the bottom. Placing the negative electrode at the bottom would drive all proteins upward into the buffer.

• Bromophenol blue dye (with some glycerol) is added on top of the IEF gel to track migration. Stop electrophoresis when the dye approaches the bottom of the gel. The dye front may curve slightly due to non-uniform salt concentration and electric field, but this does not matter.
• If the dye runs off the gel, some proteins may also have been lost, leading to incomplete results.
• The second dimension separates proteins by size only (small proteins migrate fastest).

22. Interpreting 2D Gels and Comparing Cell Types

• After electrophoresis, one glass plate is removed and the gel is stained to visualize protein spots.
• The result is a 2D map with hundreds to thousands of protein spots.
• Axes of separation:
  • All spots along a vertical line share the same PI (from the IEF dimension).
  • All spots along a horizontal line share the same molecular weight (from the SDS-PAGE dimension).
  • Smaller proteins appear toward the bottom of the gel.

  Reference figure · external source

• To compare cancer cells to normal cells: run one 2D gel for each cell type, stain both, and merge the images to identify differences.
• Sensitivity enhancement: Growing cells on S-35 methionine (a radioisotope) and using autoradiography (placing film on the gel in a freezer for about a week, then developing) is more sensitive than any chemical stain.
• An E. coli protein map from Lehninger was shown to illustrate the pattern of spots.

23. Closing

• The lecturer noted that four lectures' worth of normal-quarter material had been covered so far in the summer session.
• The next lecture will introduce microcalorimetry as the third and last technique for examining overall cellular metabolism.