"The Secret Life of Proteins: How We Clean and Use Them in Biotech"

Proteins are everywhere in your body, in food, and even in the medicines you take. But before these powerful molecules can work their magic in biotechnology, they must go through a special journey: purification. This process transforms proteins from a tangled mess inside cells into clean, functional tools ready to heal, diagnose, and build.

Let’s explore the step-by-step story of how proteins are purified and prepared for use in biotechnology without getting too technical.

What Is Protein Purification?

Protein purification is like finding a diamond in the rough. Proteins are made inside cells, often mixed with thousands of other molecules. To use them in medicine or research, scientists must separate the protein they want from everything else.

This is done through a series of steps that remove unwanted parts, leaving behind only the target protein pure and ready to use.

Step 1: Breaking Open the Cells

The first step is to release the protein from the cell. Think of it like opening a juice pouch you need to get the good stuff out!

How it’s done:

• Grinding or shaking the cells (mechanical methods)
• Using detergents or enzymes to dissolve the cell wall (chemical/enzymatic methods)

Once the cells are opened, the protein is floating in a mix of cell parts and other junk.

Step 2: Cleaning the Mixture

Now we have a messy soup of proteins, DNA, fats, and debris. It’s time to clean it up.

Cleaning techniques:

• Centrifugation – Spins the mixture to separate heavy bits from liquid.
• Filtration – Uses special filters to remove solid waste.
• Salting out (precipitation) – Adds salt to make proteins clump together so they can be collected.

This step concentrates the protein and removes the biggest impurities.

Step 3: Separating the Protein from Others (Chromatography)

Even after cleaning, we still have a mix of many proteins. Now it’s time to separate your target protein from the rest using a technique called chromatography.

Common methods:

• Ion Exchange Chromatography – Separates based on electric charge.
• Size Exclusion Chromatography – Separates based on size (big ones come out first).
• Affinity Chromatography – Uses a “lock and key” approach to grab only your protein.
• Hydrophobic Chromatography – Sorts proteins based on how oily (hydrophobic) they are.

Each method acts like a filter that chooses only the protein you need.

Step 4: Getting the Protein Ready to Use

Now that your protein is separated, it may still be in the wrong liquid or too diluted. This step prepares the protein for storage or use.

How it’s done:

• Ultrafiltration – Removes extra water or small molecules.
• Dialysis – Swaps the liquid (buffer) for one that’s better for storage.

Think of it as the final rinse before bottling.

Step 5: Testing the Protein

Before using the protein, scientists test it to make sure it's pure and works as expected.

Common tests:

• Gel tests (SDS-PAGE) – Check size and purity.
• Activity tests – Make sure the protein actually works.
• Mass spectrometry – Confirms the exact structure.
• Endotoxin testing – Ensures it’s safe for medical use.

Only after passing these tests is the protein ready for use in research or medicine.

Why It Matters

Protein purification may sound like a lab chore, but it’s a critical step in making:

• Insulin for diabetes
• Antibodies for COVID-19 and cancer
• Enzymes for food, detergents, and industry

Without purification, these proteins would be useless or even dangerous.

The Future of Protein Purification

Modern science is making protein purification faster, smarter, and greener with tools like:

• Automated machines (FPLC systems)
• AI for process optimization
• Magnetic nanoparticles for quicker separation
• Miniaturized microfluidic systems

These technologies are helping scientists produce more proteins in less time and with less waste.

 

Immunology and Immune Dysregulation

The immune system is a complex network of cells, tissues, and organs that work together to defend the body against harmful invaders like bacteria, viruses, and cancer cells. It can be broadly divided into two branches: the innate immune system and the adaptive immune system. Both systems work in concert, providing protection through a series of well-coordinated responses. However, when these systems become dysregulated, it can lead to a variety of diseases, ranging from allergies to autoimmune conditions and emerging viral diseases such as long COVID.

1. Key Concepts in Immunology

Innate and Adaptive Immunity

According to Moser and Leo (2010), the innate immune system is the body's first line of defense. It includes physical barriers such as the skin and mucous membranes, as well as cells like phagocytes (e.g., neutrophils, macrophages) that recognize and engulf pathogens. This system responds rapidly but in a non-specific manner. The adaptive immune system, on the other hand, is more specific and tailored to the pathogen at hand. It involves the activation of T cells (which help orchestrate immune responses) and B cells (which produce antibodies). The adaptive immune response also has the ability to form immune memory, ensuring that the body can mount a faster and stronger response to subsequent infections by the same pathogen.

Moser and Leo (2010) further elaborate on the coordinated actions between these two immune branches. The innate immune system not only fights pathogens but also alerts the adaptive immune system to the presence of threats. For example, the release of cytokines from infected cells or immune cells (like macrophages) signals the activation of T and B cells, which then target the pathogen specifically.

2. Immune Dysregulation in Disease

Hypersensitivity and Autoimmunity

While the immune system is essential for protection, it can also malfunction, leading to diseases. Hypersensitivity reactions, as described by Marshall et al. (2018), occur when the immune system overreacts to harmless substances. These reactions are classified into four types (Type I to Type IV), each with a different underlying mechanism. For example, Type I hypersensitivity involves IgE antibodies and is responsible for allergic reactions like hay fever, asthma, and anaphylaxis. In contrast, Type IV hypersensitivity is mediated by T cells and is associated with diseases like contact dermatitis and tuberculin sensitivity.

Autoimmunity represents another form of immune system dysfunction where the immune system mistakenly attacks the body's own tissues. The failure to maintain immune tolerance leads to the production of autoantibodies, which target the body’s own cells. As discussed by Abbas and Janeway (2000), diseases such as rheumatoid arthritis, multiple sclerosis, and type 1 diabetes are driven by such autoimmune responses. The authors highlight that the mechanisms behind autoimmunity are complex and multifactorial, involving genetic predispositions and environmental triggers that alter immune function.

3. Immune Responses in Emerging Diseases: Long COVID

In recent years, the field of immunology has had to confront the challenges posed by emerging diseases such as COVID-19. Altmann et al. (2023) provide a detailed review of the immune responses seen in long COVID, a condition in which patients continue to experience symptoms like fatigue, cognitive impairment, and respiratory issues long after the acute phase of infection.

The authors describe how immune dysregulation plays a central role in the development of long COVID. Persistent inflammation, due to ongoing activation of monocytes and macrophages, contributes to the prolonged symptoms. These cells secrete high levels of pro-inflammatory cytokines such as IL-6, which can cause chronic fatigue and muscle pain. Furthermore, T cell exhaustion, a phenomenon where T cells become dysfunctional after prolonged activation, has been observed in long COVID patients, leading to an inability to resolve the infection or clear viral remnants from the body.

Another mechanism proposed by Altmann et al. (2023) is viral persistence. They hypothesize that small amounts of the SARS-CoV-2 virus or its components may remain in the body after the acute infection resolves, triggering ongoing immune responses. Additionally, the authors discuss the role of latent viral reactivation (e.g., Epstein-Barr virus, herpesviruses) in exacerbating symptoms, as these viruses can become reactivated under conditions of immune suppression or dysregulation.

4. Clinical Applications of Immunology

The understanding of immunology has had profound implications for clinical medicine, particularly in the development of therapies and vaccines. Immunotherapy in cancer treatment, for example, has revolutionized the way oncologists approach tumor eradication. As discussed by Marshall et al. (2018), therapies like CAR-T cell therapy have shown great promise in treating cancers by genetically modifying T cells to recognize and attack cancer cells. Similarly, checkpoint inhibitors (e.g., PD-1/PD-L1 inhibitors) that block immune evasion mechanisms used by tumors have also provided significant benefits to cancer patients.

In the context of infectious diseases, vaccines are one of the greatest achievements of immunology. Warren (2016) emphasizes the role of vaccines in providing long-lasting immunity against diseases like polio, measles, and more recently, COVID-19. The development of mRNA vaccines for COVID-19 has been a major breakthrough, enabling a rapid response to the pandemic. These vaccines work by instructing cells to produce a viral protein (in this case, the spike protein of SARS-CoV-2), which triggers an immune response and provides protection against future infection.

5. Conclusion

Immunology is an ever-evolving field that is central to understanding and treating a wide range of diseases. As demonstrated by the reviewed articles, both innate and adaptive immunity play crucial roles in defending the body against infections and malignancies. However, when these systems malfunction, they can contribute to diseases such as allergies, autoimmune disorders, and long COVID.

Long COVID has brought to light the importance of understanding how the immune system can become dysregulated in the aftermath of an infection. The persistent inflammatory responses and T cell dysfunction observed in long COVID patients highlight the need for new treatment strategies targeting immune modulation. Similarly, advancements in immunotherapy and vaccines are transforming the treatment landscape for cancer and infectious diseases.

As research continues to uncover the complexities of the immune system, there is immense potential for developing targeted therapies that address the underlying immune dysfunction in a variety of diseases, from autoimmunity to chronic infections like long COVID.

References:

• Abbas, A. K., & Janeway, C. A. (2000). Immunology: Improving on nature in the twenty-first century. Cell, 100(1), 129–138.

• Altmann, D. M., Whettlock, E. M., Liu, S., Arachchillage, D. J., & Boyton, R. J. (2023). The immunology of long COVID. Nature Reviews Immunology, 23(10), 618–634.

• Marshall, J. S., Warrington, R., Watson, W., & Kim, H. L. (2018). An introduction to immunology and immunopathology. Allergy, Asthma & Clinical Immunology, 14, 1–10.

• Moser, M., & Leo, O. (2010). Key concepts in immunology. Vaccine, 28, C2-C13.

• Warren, L. (2016). Review of Medical Microbiology and Immunology. McGraw-Hill Education, 14th Edition.

Thin Layer Chromatography (TLC)

Thin Layer Chromatography (TLC) – A Simple and Colorful Tool for Compound Separation

In the world of chemistry and biology labs, Thin Layer Chromatography (TLC) is a quick and simple technique used to separate and identify compounds in a mixture. Though basic, it's powerful and widely used in research labs, pharmaceutical testing, plant studies, and even in forensics.

Let’s break down what TLC is, how it works, and why it’s so useful—step by step.

What is TLC?

Thin Layer Chromatography (TLC) is an analytical technique used to separate non-volatile mixtures. It’s especially helpful in identifying compounds like alkaloids, flavonoids, essential oils, and synthetic drugs.

The best part? It's inexpensive, fast, and you don’t need complex machinery.

Principle of TLC

TLC works on the principle of adsorption. Different compounds in a mixture stick to a surface (the stationary phase) to different extents. The more a compound sticks, the slower it moves.

Meanwhile, a solvent (mobile phase) travels up the surface by capillary action and carries the compounds along. The compounds separate because each travels at a different speed.

Basic Components of TLC

Here’s what you need for a TLC experiment:

1. TLC Plate
   A small plate made of glass, plastic, or aluminum, coated with a thin layer of adsorbent material like silica gel or alumina (this is the stationary phase).
2. Sample Mixture
   The mixture you want to separate (e.g., plant extract, dye, or drug compound).
3. Solvent (Mobile Phase)
   A liquid or mixture of liquids that moves up the TLC plate. Also called the eluent or developing solvent.
4. Developing Chamber
   A closed container with a small amount of solvent to saturate the atmosphere.
5. Capillary Tube or Spotter
   Used to apply the sample to the plate in small dots.

Step-by-Step Procedure

Here’s how TLC is performed:

1. Preparation
   Draw a pencil line 1 cm from the bottom of the TLC plate. Spot the sample on this line using a capillary tube.
2. Development
   Place the plate in the chamber with solvent. Make sure the solvent level is below the spots.
3. Separation
   As the solvent moves up, it carries the compounds. Different compounds move at different rates, forming separate spots.
4. Visualization
   After development, the spots are often invisible. Use UV light, iodine vapors, or chemical spray reagents to visualize them.
5. Calculation of Rf Value
   Measure how far each spot traveled compared to the solvent front using this formula:

Rf = Distance moved by compound / Distance moved by solvent

Interpreting TLC Results

• Rf values help identify compounds by comparing them with known standards.
• Similar Rf values = likely the same compound
• More than one spot = mixture
• No movement = strong interaction with stationary phase

Applications of TLC

TLC is used in many fields:

• Plant Chemistry – To analyze phytochemicals.
• Pharmaceuticals – To test purity of drugs.
• Food Industry – To detect additives or contamination.
• Forensics – To identify drugs, inks, or poisons.
• Organic Synthesis – To check reaction progress.

Advantages of TLC

• Fast and simple
• Inexpensive
• Needs little sample
• Can separate multiple samples at once
• Good for heat-sensitive compounds

Limitations

• Not suitable for volatile substances
• Semi-quantitative (can’t give exact concentrations)
• Requires comparison standards
• Results can vary with humidity, temperature, or solvent

 

High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography, commonly known as HPLC, is a powerful technique used in laboratories around the world to separate, identify, and quantify components in a mixture. Whether it’s used in pharmaceuticals, food industries, environmental analysis, or plant research, HPLC plays a key role in modern science.

In this article, we will explain:

• What HPLC is
• How it works (with a diagram)
• Types of HPLC
• Applications
• Advantages and Limitations

What is HPLC?

HPLC stands for High-Performance Liquid Chromatography. It is an analytical technique used to separate and analyze compounds that are dissolved in a liquid.

Basic Principle of HPLC

HPLC works on the principle of separation based on the interactions of molecules with two phases:

• Stationary Phase: A solid or liquid fixed inside a column.
• Mobile Phase: A liquid solvent that flows through the column.

Different compounds in the sample move through the column at different speeds depending on their affinity (interaction) with the stationary and mobile phases. This leads to separation.

HPLC Diagram and Components

Here is a simple diagram of an HPLC system:

Main Components of HPLC:

1. Solvent Reservoir – Contains the mobile phase (liquid solvent).
2. Pump – Pushes the mobile phase through the system.
3. Injector – Introduces the sample into the mobile phase.
4. Column – Filled with stationary phase material where separation occurs.
5. Detector – Detects the separated compounds.
6. Data System (Computer) – Displays and analyzes the results as a graph called a chromatogram.

Types of HPLC

There are different types of HPLC based on the type of stationary and mobile phases used:

1. Normal Phase HPLC
• Stationary phase: polar (e.g., silica)
• Mobile phase: non-polar (e.g., hexane)
• Used for separating polar compounds.
2. Reverse Phase HPLC (RP-HPLC)
• Stationary phase: non-polar (e.g., C18 column)
• Mobile phase: polar (e.g., water, methanol)
• Most commonly used type in pharmaceuticals and plant research.
3. Size-Exclusion HPLC
• Separates molecules based on size.
• Large molecules elute first, small ones later.
4. Ion-Exchange HPLC
• Separates based on charge (ions).
• Useful for amino acids and proteins.

What is a Chromatogram?

A chromatogram is the output of an HPLC analysis. It looks like a graph with peaks. Each peak represents a different compound, and:

• The retention time (Rt) tells us when the compound came out of the column.
• The area under the peak tells us how much of that compound is present.

Applications of HPLC

HPLC is used in many fields:

• Pharmaceutical Industry: To check drug purity and content.
• Plant Biotechnology: To identify and quantify plant metabolites.
• Food Industry: To detect preservatives, sugars, and nutrients.
• Environmental Testing: To monitor pollutants in water or soil.
• Clinical Research: To measure biomarkers in blood or urine.

Advantages of HPLC

• High accuracy and precision
• Fast analysis
• Can handle complex mixtures
• Works for heat-sensitive samples (unlike gas chromatography)

 Limitations of HPLC

• Equipment is expensive
• Requires trained personnel
• Uses a lot of solvents (which may be toxic)
• Regular maintenance need

 

FTIR- Fourier Transform Infrared Spectroscopy

Principle, Methods, Applications, Tools, and Chemical Identification

1. Principle of FTIR

Fourier Transform Infrared Spectroscopy (FTIR) is an analytical technique used to identify organic, polymeric, and, in some cases, inorganic materials. The principle is based on the absorption of infrared radiation by a sample, resulting in vibrational transitions in molecular bonds.

• IR Radiation is passed through a sample.
• Molecules absorb specific frequencies of IR light that match their natural vibrational frequencies.
• The absorption pattern (spectra) is like a molecular fingerprint.
• The interferogram is converted into a spectrum using a Fourier Transform algorithm.

2. Methods of FTIR

There are two main modes of FTIR analysis:

a. Transmission Mode

• Sample is placed directly in the path of the IR beam.
• Common for liquids and transparent solids.

b. Attenuated Total Reflectance (ATR)

• IR light reflects internally through a crystal in contact with the sample.
• Ideal for solids, powders, and films without preparation.

c. Diffuse Reflectance (DRIFTS)

• Suitable for powdered or rough-surfaced samples.
• IR beam is reflected and scattered by the sample.

d. Reflectance/Specular Reflectance

• Used for thin films or surface coatings.

3. Applications of FTIR

FTIR is widely applied in several fields:

• Pharmaceuticals: Identifying functional groups in drug compounds.
• Polymers: Analyzing polymer structures and degradation.
• Environmental Science: Detecting pollutants or contaminants.
• Food Industry: Identifying food adulterants or quality testing.
• Nanotechnology: Monitoring surface modifications and nanoparticle functionalization.
• Botany and Biotech: Identifying phytochemicals and metabolites in plant extracts or callus culture.

4. Tools and Components Used in FTIR

• IR Source: Typically, a Globar (silicon carbide) or Nernst glower.
• Interferometer: Michelson interferometer to modulate the IR signal.
• Beam Splitter: Divides the IR beam (commonly made of KBr or CaF₂).
• Sample Holder/Stage: Holds solid, liquid, or powdered samples.
• Detector: Converts IR signal to an electrical signal (DTGS or MCT detectors).
• Computer + Software: For Fourier transform and spectrum analysis.

5. Common Chemical Names Detected in FTIR

FTIR identifies functional groups by detecting bond vibrations. Examples:

Functional Group	Wavenumber (cm⁻¹)	Typical Compounds Detected
–OH (hydroxyl)	3200–3600	Alcohols, Phenols
–NH (amine)	3300–3500	Amines, Proteins
C=O (carbonyl)	1650–1750	Aldehydes, Ketones, Esters
–CH (alkane/alkene)	2800–3100	Hydrocarbons
–COOH (carboxylic)	1700–1725 & 2500–3300	Carboxylic acids
–C≡C, –C≡N	2100–2260	Alkynes, Nitriles
–C–O–C– (ether)	1000–1300	Ethers, Glycosides

These are often used to identify:

• Flavonoids
• Phenolics
• Alkaloids
• Terpenoids
• Carbohydrates
• Proteins/Peptides

FTIR Procedure

To launch the FTIR program, double-click the OMNIC icon.

⬇️
From the Collect pull-down menu, select Setup, and select the following:

• No. of scans = 8
• Resolution = 2 cm⁻¹
• Zero filling = None
• Final format = Absorbance

⬇️
From the Edit pull-down menu, select Options. Click on the Collect tab at the top. Click the “Collect to New Window” box to deselect it. Click OK to close the window.

⬇️
Select Background from the Collect pull-down menu (Repeat background collection every 30 minutes to 1 hr. if the humidity is high).

⬇️
From the Collect pull-down menu, select Collect. Sample: Place the sample in the holder in the IR, then select Yes. Add to window = Yes

⬇️
From the Process pull-down menu, select: Automatic Baseline. Correct old spectrum shown in red and the new spectrum shown in blue. Click on the red one. From the Edit pull-down select Clear from the Process pull-down menu, select % Transmittance.

⬇️
From the View pull-down menu, select Display Limits (to change the axes of the graph).

⬇️
During spectrum display, click on T (for text) at the bottom of the page. Click on peaks of interest to label.

⬇️
From the File pull-down menu, select Print.

⬇️
To Run the Next Sample: from the Window pull-down menu, select New Window (more than 7 open windows will crash OMNIC).

⬇️
Repeat steps 4–8 above.

KBr Pellet Procedure for Solid Samples

• Take about 1/8th of the solid sample on a micro spatula and about 0.25–0.50 teaspoons of KBr.
• Mix thoroughly in a mortar while grinding with the pestle. If the sample is in large crystals, grind the sample separately before adding KBr.
• Place just enough spl. to cover the bottom in the pellet die.
• Place in press and press at 5000–10000 psi.
• Carefully remove the pressed sample from the die and place it in the FTIR sample holder.
• The pressed disc should be nearly clear if properly made. If it is translucent, regrind and repress.