Protein Sequencing Techniques and PCR

### Electrophoresis 1

• Electrophoresis is a technique used to separate and sometimes purify charged macromolecules that differ in size, charge and conformation in an electric field.
• When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.
• The greater the charge of a macromolecule, the faster it migrates (greater electrophoretic mobility).
• Greater size of the macromolecule will have greater frictional and electrostatic forces, slower electrophoretic mobility.
• Rounded molecules have lesser frictional and electrostatic retardation compared to non-globular structure.
• Globular proteins move faster than fibrous proteins; supercoiled plasmid move faster than linear plasmid.
• DNA (and RNA) molecules are negatively charged, when placed in an electric field, they migrate to the positively charged anode.
• DNA fragments (double-stranded linear DNA) have a uniform mass/charge ratio; thus, DNA fragments are separated by size within an agarose gel.
• Shorter fragments migrate faster, longer fragments migrate slower.
• The distance migrated on a gel correlates inversely with the logarithm of molecular weight.
• Proteins and nucleic acids migrate within a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel.
• These are solid yet porous matrixes.
• Molecules will move through the matrix at different rates, usually determined by their mass, charge and conformation; they eventually get separated as different 'bands'.
• Molecular weight markers are run side by side with samples; the molecular weight of samples can be estimated by referring to the markers.
• DNA electrophoresis often uses agarose gel.
• Protein electrophoresis often uses polyacrylamide gel.

Electrophoresis 2

• SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a technique used to resolve and separate proteins based on molecular weight.
• Proteins are mixed with Laemmli buffer before electrophoresis.
• Laemmli buffer contains SDS (an anionic detergent), 2-mercaptoethanol (breaks disulfide bonds in globular proteins), and glycerol (high density solution to pull protein sample down and stay in its designated well).
• SDS gives all proteins an overall negative charge proportional to its mass
• 2-mercaptoethanol breaks disulfide bonds in globular proteins.
• Glycerol-high density solution- pull DNA sample and settle to the bottom of the well.
• The electrophoretic migration rate through a gel is therefore determined only by the size of the protein.
• All proteins, because of the negative charge imparted by SDS, move toward the positive electrode.
• Molecular weights are determined by simultaneously running marker proteins of known molecular weight.
• Native PAGE separates proteins based on their charge, mass, and shape (conformation), without denaturing them.
• Isoelectric focusing (IEF) is an electrophoretic separation technique based on the isoelectric points (pI) of molecules.
• Proteins are loaded; they will move to anode/cathode according to their net charge.
• They will reach a point where the gel pH is equal to their pI.
• Because proteins have zero net charge when pH = pI; they cease moving.
• The protein is said to 'focus' at this point.

• A 2-D gel electrophoresis technique is used to separate protein mixtures using two different principles, which improves resolution.

• Western blotting is a method for separating and detecting specific proteins from a mixture of proteins.

• The main steps are sample preparation and electrophoresis, electrophoretic transfer, blocking, antibody incubation, and detection.

• ELISA (enzyme-linked immunosorbent assay) is an immunoassay detecting and quantifying small molecules, like antibodies, peptides, antigens or proteins.

• ELISA is a simple and sensitive technique for quantitative information output, using enzyme-conjugated antibodies or fluorescence-conjugated antibodies.

• ELISA relies on several key reagents, including antigen, antibody, enzyme-conjugated antibody, and a substrate.

• Polymerase chain reaction (PCR) is a technique for amplifying target DNA sequences.

• A thermal cycler is a laboratory instrument used to increase and decrease the temperature of the reaction mixtures in controlled, pre-programmed steps.

• PCR requires several chemicals including template DNA, primers (forward and reverse), deoxynucleotide triphosphates (dNTPs), DNA polymerase, PCR buffers, and nuclease-free water.

• Types of PCR: Conventional, Real-time, and Digital.

• Real-time PCR allows real-time monitoring of the newly generated PCR product during the process by using fluorescence labelled oligonucleotide

• DNA ladder is used as a molecular marker, which is a set of DNA standards with known base pair sizes for identifying the approximate size of the DNA sample during electrophoresis.

• DNA amplified is visible using fluorescent dye, like Ethidium bromide.

• Capillary electrophoresis is a method of separating charged macromolecules by their charge-to-mass ratio

• Capillary electrophoresis also refers to the time required for molecules to travel through a matrix.

### Other Techniques

•  Some additional scientific techniques include (but are not limited to) the following: oligonucleotide/peptide synthesis, microarray (DNA or protein) and centrifuge.

### Centrifuge 1

•  A centrifuge is a device to separate particles from a solution by size, shape and density.

•  The principle of centrifugation is based on the centrifugal force generated during high-speed spinning that makes particles, with a greater density, to settle towards the bottom of a solution faster.

•  Relative centrifugal force (RCF) is a transferable unit used across manufacturers’ centrifuges.

•  Nomograms translate revolutions per minute (RPM) to relative centrifugal force (RCF) or vice versa.

### Centrifuge 2

•  Types of centrifugation methods include:
•  Cell fractionation, involving the disruption and lysis of a cell membrane via homogenization
•  Differential centrifugation, isolating different cellular components by separating them via successive cycles of high-speed centrifugation,
•  Gradient centrifugation, separating substances based on their density and sedimentation rate in a density gradient

Microscopy 1

•  A microscope is a laboratory instrument used to examine small objects or specimens not viewable by the naked eye.

•  The history of the microscope traces back to the Dutch Janssen brothers who invented the first microscope in 1590, further developed by Antonie van Leeuwenhoek

•  Types of microscopes

•  Light Microscopy (includes simple and compound microscopes, brightfield, darkfield, phase contrast)

•  Electron Microscopy (transmission and scanning)

Microscopy 2

•  Types of Light Microscopes: These include Simple, Compound, Bright Field, Dark Field, Phase Contrast, Confocal, and Polarized. 

•  Working principles, components, and functions for each type. 

•  Applications & Advantages

•  Limits & Usefulness

### Other Techniques

•  A few added scientific techniques include (but are not limited to) the following, flow cytometry, Reverse Transcription & Polymerase Chain Reaction

### Equipment

•  Centrifuge

•  Microscope (includes different components like lenses, optics)

•  Various kinds of equipment for use in scientific research labs.