Lec 6/7 (App 3) Protein Stability Measuring Protein Concentration/Calculating Cell Numbers (Biomass

1. State why denaturation does not affect a protein’s primary structure.

Denaturation breaks bonds that hold the protein’s shape.

The primary structure is just the sequence of amino acids.

That sequence is held by strong peptide bonds, which are not broken during denaturation.

🔹 2. State the types of bonds that are disrupted when a protein is exposed to adverse temperature and pH.

Hydrogen bonds

Ionic bonds

Hydrophobic interactions

Disulfide bonds (in some cases)

These bonds hold the protein’s shape, and changes in heat or pH can weaken or break them.

🔹 3. Describe how oxidation and reduction can affect protein stability through redox reactions.

Proteins have disulfide bonds that help keep their shape.

Oxidation can create disulfide bonds (S–S), stabilizing the protein.

Reduction breaks these bonds, possibly causing unfolding or loss of structure.

🔹 4. Explain in words and by drawing a diagram, how SDS and β-mercaptoethanol affect protein structure, and highlight how this might affect SDS electrophoresis during recombinant production purification.

SDS (sodium dodecyl sulfate): Breaks non-covalent bonds and coats the protein with negative charges. This gives all proteins a similar charge-to-size ratio.

β-mercaptoethanol: Breaks disulfide bonds between cysteines, separating protein subunits.

This makes all proteins unfold and move only by size in SDS-PAGE, helping you check protein purity during recombinant production

5. Explain how stability affects proteins and explain why this must be preserved for therapeutic products.

Stable proteins keep their shape and function.

Unstable proteins can unfold, lose activity, or break down.

For therapeutic proteins, stability is vital for safety, correct function, and long shelf-life.

🔹 6. Explain why protein absorbance is measured at 280 nm and explain limitations of this method.

Proteins absorb light at 280 nm due to tryptophan and tyrosine.

It's quick and does not need extra reagents.

Limitations:

Not accurate if other UV-absorbing molecules (like DNA) are present.

Doesn’t tell you exact protein concentration if the amino acid content varies.

🔹 7. Compare and contrast the reactions that underlie the Lowry and Bradford protein assays, highlighting the pH requirements, reagents, and absorbance values for each.

Feature Lowry Assay Bradford Assay

Reaction Protein + copper → color reaction with Folin reagent Protein + Coomassie dye → dye binds to protein

pH Alkaline (pH ~10) Acidic (pH ~1)

Reagents Copper sulfate, Folin–Ciocalteu reagent Coomassie Brilliant Blue G-250

Absorbance 750 nm 595 nm

Sensitivity More sensitive, but complex Simple and fast, less sensitive

🔹 8. Describe the principles and key steps of an immunohistochemistry experiment.

Principle:

Detect specific proteins in tissue using antibodies linked to a color or fluorescent label.

Steps:

1. Fix the tissue to preserve it.

2. Block non-specific binding sites.

3. Add a primary antibody (binds the target protein).

4. Add a secondary antibody (linked to a label and binds the primary).

5. Add a substrate that reacts with the label to produce color or fluorescence.

6. View under a microscope.

🔹 9. Construct a workflow of measuring protein levels in a sample using SDS-PAGE / Western Blot techniques.

SDS-PAGE / Western Blot Workflow:

1. Sample Prep: Lyse cells and collect protein.

2. Add SDS + β-mercaptoethanol to denature proteins.

3. Run SDS-PAGE to separate proteins by size.

4. Transfer proteins to a membrane (e.g. PVDF or nitrocellulose).

5. Block membrane to prevent non-specific binding.

6. Add primary antibody (binds your protein of interest).

7. Add secondary antibody (has enzyme or label).

8. Detect signal using chemiluminescence or color reaction.

9. Analyze band intensity to measure protein levels.

(c) Western Blotting (13 marks)

Western blotting is a technique used to detect specific proteins in a sample.

Steps:

1. Sample Preparation: Proteins are extracted from cells or tissues.

2. SDS-PAGE: Proteins are denatured and separated by size using gel electrophoresis.

3. Transfer: Proteins are transferred from the gel to a membrane (e.g., nitrocellulose or PVDF).

4. Blocking: Membrane is blocked with a protein solution to prevent nonspecific binding.

5. Primary Antibody Incubation: Specific antibody binds to the target protein.

6. Secondary Antibody Incubation: An enzyme-linked antibody binds the primary antibody.

7. Detection: Substrate is added. Enzyme converts substrate to a detectable signal (chemiluminescence or color change).

Applications:

Detecting protein expression levels.

Confirming the presence of pathogens (e.g., HIV).

Checking for post-translational modifications.

Diagram Suggestion: A labeled flow chart showing:

SDS-PAGE → Transfer → Block → Primary Ab → Secondary Ab → Detection

🔹 10. Why is measuring cell number or biomass important in biotechnology?

It helps track cell growth during fermentation or bioprocessing.

Ensures optimum yield of products like proteins or enzymes.

Useful for monitoring health and productivity of cultures.

Helps decide when to harvest cells or adjust conditions (pH, nutrients, etc.).

🔹 11. Outline procedures for measuring biomass by wet/dry weight and plate counting.

Wet Weight:

Harvest cells by centrifugation.

Remove the liquid.

Weigh the wet cell pellet.

Dry Weight:

After centrifugation, dry the cell pellet in an oven (usually at 80–100°C).

Weigh the dry residue.

Plate Counting:

Dilute the sample in steps (e.g. 1:10, 1:100...).

Plate a known volume on agar plates.

Incubate and count the number of colonies formed.

Multiply by dilution factor to get colony-forming units per mL (CFU/mL).

🔹 12. Compare and contrast biomass measurement methods

Method Advantages Disadvantages

Wet Weight Fast, simple Includes water weight, less accurate

Dry Weight More accurate biomass measurement Takes longer, needs drying equipment

Optical Density (e.g. OD600) Quick and easy, no plating needed Measures total cells (dead + alive), not exact count

Tetrazolium Test Measures live (active) cells Needs reagents and sometimes special equipment

Plate Counting Measures only viable cells Slow (24–48 hours), assumes 1 colony = 1 cell

🔹 13. Define viable cell density (in words and equation)

In Words:

Viable cell density is the number of living (capable of dividing) cells in a given volume of culture.

Equation:

Viable Cell Density = (Number of colonies × dilution factor) / volume plated

Usually expressed as CFU/mL (colony-forming units per millilitre).

🔹 14. Recent advances in measuring biomass using electrical and fluorescent indicators

Electrical Impedance Sensors:

Measure changes in electrical current caused by cell presence.

More accurate for detecting live cells in real time.

Fluorescent Probes:

Use dyes that only enter live cells or react with certain enzymes.

Common dyes: Calcein-AM (live cells), Propidium iodide (dead cells).

Used in flow cytometry or fluorescence microscopes.

Benefits:

Faster, more sensitive.

Can detect small changes in growth.

Good for high-throughput screening in biotechnology labs.

🔹 15. Principle of using tetrazolium indicators for viable cell counts

Living cells have active enzymes that reduce tetrazolium salts.

This reduction forms a colored product.

Example:

Tetrazolium salt (like MTT) is reduced by dehydrogenase enzymes (from active mitochondria).

The product is formazan—a purple, insoluble compound.

The amount of formazan reflects the number of viable cells.