Blotting Techniques

Overview

Definition

Blotting is an investigative technique used to detect and identify macromolecules, such as nucleic acids Acids Chemical compounds which yield hydrogen ions or protons when dissolved in water, whose hydrogen can be replaced by metals or basic radicals, or which react with bases to form salts and water (neutralization). An extension of the term includes substances dissolved in media other than water. Acid-Base Balance and proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis. This technique is accomplished by:

• Separation using electrophoresis
• Transfer to a membrane
• Detection with a labeled probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography)

Types of blotting

The 4 basic types of blotting are

• Southern
• Northern
• Western
• Eastern (rarely used in clinical setting)

Comparison of blotting techniques

Electrophoresis

All blotting techniques use electrophoresis, which involves using an electrical field to separate molecules. 

• Molecules are separated by:
  • Size
  • Electrical charge
• Used extensively in analysis of:
  • DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure
  • RNA RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. RNA Types and Structure
  • Proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis
• Types:
  • Cataphoresis: electrophoresis of cations Cations Positively charged atoms, radicals or groups of atoms which travel to the cathode or negative pole during electrolysis. Electrolytes  
  • Anaphoresis: electrophoresis of anions Anions Negatively charged atoms, radicals or groups of atoms which travel to the anode or positive pole during electrolysis. Electrolytes
• Technique:
  • Sample is loaded onto a gel.
  • Gel is placed in a buffer solution.
  • An electric field is applied.
  • The electrical current Electrical current The flow of charged particles from one point to another (in physiology, usually across a cell membrane) Cardiac Physiology separates the molecules.

Procedure

General blotting technique

Blotting techniques follow a general procedure:

• The target molecule in a sample is isolated.
• Electrophoresis separates the molecules.
• The separated contents are transferred (or blotted) onto a filter or membrane to produce a  “print.”
• The filter/membrane is then exposed to radiolabeled probes and incubated → probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) binds to the target molecule
• The probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) and target molecule create bands that can be visualized with X-ray X-ray Penetrating electromagnetic radiation emitted when the inner orbital electrons of an atom are excited and release radiant energy. X-ray wavelengths range from 1 pm to 10 nm. Hard x-rays are the higher energy, shorter wavelength x-rays. Soft x-rays or grenz rays are less energetic and longer in wavelength. The short wavelength end of the x-ray spectrum overlaps the gamma rays wavelength range. The distinction between gamma rays and x-rays is based on their radiation source. Pulmonary Function Tests film.
• Note: probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) and detection methods can also use:
  • Colorimetry
  • Chemiluminescence

Southern blotting

• Restriction enzymes Enzymes Enzymes are complex protein biocatalysts that accelerate chemical reactions without being consumed by them. Due to the body’s constant metabolic needs, the absence of enzymes would make life unsustainable, as reactions would occur too slowly without these molecules. Basics of Enzymes are needed to cleave the DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure sample into fragments prior to electrophoresis
• Prior to blotting, the separated double-stranded DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure are denatured with alkaline solution → single-stranded DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure fragments
• Blotting method: capillary transfer of DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure from an electrophoresis gel to a filter
• Uses labeled oligonucleotide probes that are complementary to the target DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure sequence
• Incubation Incubation The amount time between exposure to an infectious agent and becoming symptomatic. Rabies Virus allows hybridization of the probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) with the target DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure.

Northern blotting

• Blotting method: capillary transfer of RNA RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. RNA Types and Structure from an electrophoresis gel to a filter
• Uses labeled oligonucleotide probes that are complementary to the target RNA RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. RNA Types and Structure sequence
• Incubation Incubation The amount time between exposure to an infectious agent and becoming symptomatic. Rabies Virus allows hybridization of the probe Probe A device placed on the patient’s body to visualize a target Ultrasound (Sonography) with the target RNA RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. RNA Types and Structure.

Western blotting

• Sample proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis are applied to an electrophoresis gel. (In certain situations, the proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis must be denatured before they are applied.)
• Blotting method: Proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis are electrophoretically transferred (using an electric field) from the gel to the blotting membrane.
• Uses antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins: Types and Functions that are specific to the target protein; this antibody may be:
  • Unlabeled or primary (most common) → if so, a secondary, labeled antibody that can bind BIND Hyperbilirubinemia of the Newborn to the primary antibody is then used
  • Labeled (less common)
• Incubation Incubation The amount time between exposure to an infectious agent and becoming symptomatic. Rabies Virus allows antibody– antigen Antigen Substances that are recognized by the immune system and induce an immune reaction. Vaccination complexes to form.

Uses

Southern blot

• Can detect:
  • Small and large DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure mutations:
    • Deletions
    • Duplications
    • Rearrangements
  • Gene Gene A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. Basic Terms of Genetics methylation Methylation Addition of methyl groups. In histo-chemistry methylation is used to esterify carboxyl groups and remove sulfate groups by treating tissue sections with hot methanol in the presence of hydrochloric acid. . Glucocorticoids changes
• Applications:
  • Forensics Forensics Disciplines that apply sciences to law. Forensic sciences include a wide range of disciplines, such as forensic toxicology; forensic anthropology; forensic medicine; forensic dentistry; and others. Polymerase Chain Reaction (PCR)
  • Identifying new disease-causing mutations
  • Diagnosis of genetic conditions
  • Identification Identification Defense Mechanisms of malignancy-specific genetic markers (e.g., BCR-ABL1 gene Gene A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. Basic Terms of Genetics fusion in CML CML Chronic myeloid leukemia is a malignant proliferation of the granulocytic cell line characterized by a fairly normal differentiation. The underlying genetic abnormality is the Philadelphia chromosome, an abbreviated chromosome 22, resulting from reciprocal (9;22)(q34;q11) translocation. Chronic Myeloid Leukemia)

Northern blot

Northern blotting can be used to assess RNA RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. RNA Types and Structure levels and gene Gene A category of nucleic acid sequences that function as units of heredity and which code for the basic instructions for the development, reproduction, and maintenance of organisms. Basic Terms of Genetics expression.

Western blot

Identification Identification Defense Mechanisms of antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins: Types and Functions:

• Diagnosis of infectious diseases, including:
  • HIV HIV Anti-HIV Drugs 
  • Lyme disease Lyme disease Lyme disease is a tick-borne infection caused by the gram-negative spirochete Borrelia burgdorferi. Lyme disease is transmitted by the black-legged Ixodes tick (known as a deer tick), which is only found in specific geographic regions. Patient presentation can vary depending on the stage of the disease and may include a characteristic erythema migrans rash. Lyme Disease
  • Rickettsia Rickettsia Rickettsiae are a diverse collection of obligate intracellular, gram-negative bacteria that have a tropism for vascular endothelial cells. The vectors for transmission vary by species but include ticks, fleas, mites, and lice. Rickettsia infections Infections Invasion of the host organism by microorganisms or their toxins or by parasites that can cause pathological conditions or diseases. Chronic Granulomatous Disease
• Identification Identification Defense Mechanisms of abnormal proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis and antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins: Types and Functions, including:
  • Prion disease
  • Muscular dystrophy Muscular Dystrophy Becker Muscular Dystrophy (dystrophin analysis)
  • Autoantibodies (e.g., anti–glomerular basement membrane Basement membrane A darkly stained mat-like extracellular matrix (ecm) that separates cell layers, such as epithelium from endothelium or a layer of connective tissue. The ecm layer that supports an overlying epithelium or endothelium is called basal lamina. Basement membrane (bm) can be formed by the fusion of either two adjacent basal laminae or a basal lamina with an adjacent reticular lamina of connective tissue. Bm, composed mainly of type IV collagen; glycoprotein laminin; and proteoglycan, provides barriers as well as channels between interacting cell layers. Thin Basement Membrane Nephropathy (TBMN) (GBM) antibodies Antibodies Immunoglobulins (Igs), also known as antibodies, are glycoprotein molecules produced by plasma cells that act in immune responses by recognizing and binding particular antigens. The various Ig classes are IgG (the most abundant), IgM, IgE, IgD, and IgA, which differ in their biologic features, structure, target specificity, and distribution. Immunoglobulins: Types and Functions)

Advantages and Disadvantages

Advantages

• All kinds: good specificity
• Southern blot: can detect a wide variety of mutations
• Western blot:
  • Good sensitivity
  • Can evaluate for several target proteins Proteins Linear polypeptides that are synthesized on ribosomes and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of amino acids determines the shape the polypeptide will take, during protein folding, and the function of the protein. Energy Homeostasis

General disadvantages

• Requires a large amount of DNA DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA Types and Structure or RNA RNA A polynucleotide consisting essentially of chains with a repeating backbone of phosphate and ribose units to which nitrogenous bases are attached. RNA is unique among biological macromolecules in that it can encode genetic information, serve as an abundant structural component of cells, and also possesses catalytic activity. RNA Types and Structure
• Slow, labor-intensive process
• Expensive
• Use of radioactive materials can potentially be hazardous.