Advanced Protein and DNA Analysis Techniques: Western Blotting and Binding Assays

Properties of Proteins and DNA Visibility

• Optical Properties of Biomolecules: Generally, both DNA and proteins are clear in solution and cannot be seen with the naked eye.
• Exceptions to Protein Clarity: Proteins may appear colored if they have an organic molecule docked to them. For example, the heme group docked to certain proteins contains ring structures that affect light absorption, giving the protein a visible color.
• Necessity of Staining: Because they are generally clear, proteins and DNA must be stained to be visualized after being separated via gel electrophoresis.

Gel Electrophoresis Basics and Visualization

• Separation Mechanism: Gels act as a matrix, similar to a fishing net. Smaller molecules migrate faster through the pores and reach the bottom of the gel sooner, while larger molecules move slower.
• Molecular Weight Scaling: Separation is based on mass. If scales were sensitive enough, we could theoretically distinguish every person in the human population by their exact mass; similarly, gel electrophoresis separates proteins based on their specific molecular weights.
• Interpretation of Bands:
  • Position: Proteins migrating further/faster have a lower molecular weight ($MW$).
  • Thickness: Band thickness reflects abundance. It is a quantitative measurement; a thicker, fatter band indicates more protein is present.
• Staining Sensitivities:
  • Coomassie Blue: Not very sensitive; requires approximately a microgram ($1\,\mu g$) quantity of protein to be visible.
  • Silver Staining: Highly sensitive; can detect proteins down to nanogram ($ng$) quantities.
• Complexity of Tissues: Humans have approximately $23,000$ genes. If a tissue like the kidney expresses half of those, a stained gel would show roughly $12,000$ bands. This often appears as a "smear" of complexes, making it impossible to identify a specific "Protein X" by mass alone because many different proteins share similar weights.

Western Blotting Protocol and Methodology

• Definition: A technique to transfer proteins from a gel to a stable membrane (nitrocellulose or nylon) for identification using specific probes (antibodies).
• Transfer Mechanism: Unlike DNA (Southern blotting) or RNA (Northern blotting), which can be transferred via capillary action (e.g., using a Pyrex dish and paper towels), proteins do not easily exit the pores of the gel. They require an electric current to push them out.
• The Tank Method:
  • A "sandwich" is built consisting of the gel, nitrocellulose paper, and Whatman paper.
  • The sandwich is submerged in a buffer tank.
  • Directionality: Directionality of the flow is critical. If the current is applied in the wrong direction, the proteins will move into the buffer instead of onto the membrane.
• Membrane Characteristics: Nitrocellulose is a polysaccharide made of glucose with specific linkages and added nitro groups. It is structurally strong and binds proteins very well, creating a "print" of the gel.

Detection via Antibodies (Probing)

• Primary Antibody:
  • Acts as the specific probe for the target protein (antigen).
  • It typically recognizes a sequence of approximately $6$ to $8$ amino acids.
  • Usually produced in model organisms like mice or rabbits.
  • If targeting "Protein X," the probe is called an "anti-protein X antibody."
• Secondary Antibody:
  • Used to visualize the binding event because antibodies themselves are clear proteins.
  • Specific for the primary antibody (e.g., if the primary was made in a rabbit, the secondary is an anti-rabbit antibody).
  • Enzyme Conjugation: Secondary antibodies are covalently linked to enzymes that remain functional. Common enzymes include:
    • HRP: Horseradish Peroxidase.
    • AP: Alkaline Phosphatase.
• Visualization: A substrate is added (e.g., a colorless dye called "XP"). The enzyme converts the substrate into a detectable signal, such as a black color or light emission (chemiluminescence).

Studying Gene Expression: Protein vs. RNA

• Logic of Expression: Higher gene activity leads to more mRNA, which typically leads to more protein. Therefore, both Northern blots (RNA) and Western blots (protein) can measure gene activity.
• Northern Blotting:
  • Measures mRNA levels.
  • Uses a cDNA probe obtained via RT-PCR (Reverse Transcription Polymerase Chain Reaction).
  • General preference: Often faster and cheaper than Western blotting.
• Western Blotting:
  • Measures protein levels.
  • More difficult due to antibody production. Obtaining an antibody can take months, involving animal injections (e.g., in the large veins of a rabbit's ears) and blood testing to ensure the antibody is specific and not contaminated by other immune responses.
• Correlative Analysis: Scientists often perform both to see if RNA and protein levels correlate; approximately $90\%$ of the time, they do.

SDS-PAGE (Polyacrylamide Gel Electrophoresis)

• Matrix: Uses polyacrylamide rather than agarose.
• Toxicity: Polyacrylamide is a neurotoxin in its liquid state.
• Orientation: Unlike agarose gels, which are "submarine gels" run horizontally and submerged in buffer, protein gels (SDS-PAGE) are run vertically.
• Casting: Gels are cast between plates. Because of the vertical nature, there is a risk of leaking due to gravity before polymerization occurs.

DNA-Binding Assays: EMSA (Gel Shift)

• Names: Electrophoretic Mobility Shift Assay (EMSA), Gel Shift Assay, Gel Retardation Assay.
• Purpose: To determine if a protein can bind to a specific DNA sequence.
• Gel Conditions: Must use "native" (non-denaturing) polyacrylamide gels. If detergent or heat is used to unfold the protein, it will no longer recognize and bind to the DNA.
• Probe:
  • Double-stranded DNA (dsDNA), typically radioactive.
  • Length: Usually around $20$ base pairs ($bp$).
  • Preparation: Synthesized as two complementary strands that are annealed together (not typically made via PCR due to the short length).
• Mechanism:
  • A "shift" occurs when a protein binds to the DNA, increasing its mass and causing it to migrate slower (retardation).
  • Multiple bands represent different protein-DNA complexes.
• Super Shift: If an antibody specific to a protein (e.g., Protein X) is added and it binds the already-formed protein-DNA complex, the mass increases further, shifting the band even higher on the gel. This confirms the presence of that specific protein in the complex.
• Sample Preparation: Uses "nuclear lysates." Cells are popped using a hypotonic solution (lower salt concentration than the cell's internal $0.9\%\,NaCl$), causing them to swell and burst. The nuclei are then isolated and treated to extract transcription factors.
• Limitations: It is an in vitro assay using "naked" DNA, meaning it does not account for the natural presence of nucleosomes.

DNA-Binding Assays: DNase I Footprinting

• Purpose: To identify the exact nucleotide contact points where a protein binds to DNA.
• Probe:
  • Larger piece of DNA, approximately $150$ to $300$ base pairs ($bp$).
  • Labeling: Critical that only one end of one strand is radioactively labeled.
• Mechanism:
  • The protein complex is allowed to bind to the DNA.
  • The enzyme DNase I (a nuclease) is added at a controlled concentration to cut the DNA.
  • The protein protects the DNA section it covers from being cut.
• The "Footprint":
  • The DNA is denatured (separated into single strands) using a polyacrylamide gel containing urea.
  • A control lane (DNA without protein) shows a continuous ladder of fragments of every possible length.
  • The experimental lane (DNA + protein) will show a gap (the footprint) where no bands appear, because the protein prevented DNase I from cutting at those specific lengths.
• Technical Challenges: If the DNase I concentration is too high, it will over-digest the DNA, resulting in only very small fragments at the bottom and a loss of data.