PROTEIN ANALYSIS

• Berzelius

• Protein

In the earlier days, the complex nitrogen-rich substance found in cells of all animals and plants are not identified until ___ suggested the name “___”

Proteios = Primary

Protein came from the Greek word ___ which means ___

False.

Most of the 20 basic amino acids present in proteins were present from 1819 until 1904.

True or False.

Most of the 20 basic amino acids present in proteins were present from 1809 until 1914.

Proteins

They are the building blocks of the cell, which constitute the majority of its dry mass.

Answer: I, II, IV

III: should be nitrogen-rich substance

V: 20 basic amino acids

Which of the ff. describes proteins?

I. Product of transcription

II. Product of translation

III. Carbon-rich substance

IV. Contains the impact of data processed by nucleic acid

V. Has 21 basic amino acids

• Chromatography

• Martin and Synge (1942)

• A technique which is now widely used to separate proteins.

• This is developed by?

• Primary structure

• Secondary Structure

• Tertiary structure

• Quaternary structure

What are the different structures of proteins?

Primary Structure

This is composed of the sequence of a chain of amino acids.

Secondary structure

There is a hydrogen bonding in the peptide backbone, causing the amino acids to fold in repeating patterns.

• a-helices

• B-helices

What are the different patterns of secondary structure?

Tertiary structure

There is a 3-Dimensional folding of patterns of the protein due to the side chain interaction.

Quaternary structure

There is more than one amino acid chain.

Polymers

It is composed of covalently-linked amino acid residues and the carboxyl groups combined by peptide bonds.

Glycine

It contains a side chain of a single hydrogen.

Alanine

It contains a side chain of a methyl group.

Polypeptides

The amino acids form a long, linear chain, which is called?

Polypeptides → specific amino acid chain

It specifies the protein structure.

They share the same electrons

Peptide bonds release a water molecule because?

Proline

It doesn’t have an amino group.

Hydrogen bonds

Alpha helix and Beta sheets are held together by?

• 4th

• Twist

In alpha helix, the hydrogen bonds form in every ___ amino acid and causes a ___ in the amino acid chain.

• Pleats

• Atoms

In beta sheets, the ___ are formed by the hydrogen bond between the ___ on the backbone of the polypeptide chains.

Tertiary structure

The protein molecule will bend and twist in a way that it could achieve the maximum stability and lowest energy state.

Quaternary structure

This is the association of two or more polypeptides into a multi-subunit complex. This is the assembly of individual polypeptides into a larger, functional clusters.

Hemoglobin (4 subunits: 2 alpha, 2 beta)

An example of quaternary structure

• Sample Type

• Location of Protein of Interest

• Required Protein yield for the subsequent analysis to be performed

• Method utilized

The procedure that may be used to achieve optimal isolation of proteins/ Protein Isolation Procedures depends upon:

• Western blotting

• Enzyme-linked immunosorbent (ELISA)

• Gel shift assays (EMSA)

• Reporter Assays

• Mass Spectrometry and Immunoprecipitation

Proposed downstream applications for the quantification and identification of desired proteins.

• Natural sources such as mammalian cell cultures

• Tissues or bodily fluids

• Overexpression in a model system such as:

  • Bacteria

  • Yeast

  • Insect or mammalian cells

  • Monoclonal antibodies from hybridoma cells

  • Plant cells used in agricultural biotechnology

Sources of proteins

Overexpression

This is the process where the gene are expressed at higher levels than normal within the cell or organism.

• Naturally

• Artificially induced

Overexpression can happen:

• Produce large quantities of proteins

• Monoclonal antibodies

• Purpose of overexpression

• Example

• Mechanical disruption

• Liquid homogenization

• High-frequency sound waves

• Freezing/thaw cycles

• Manual grinding

Several methods are commonly used for physical lysis cells, including:

• Require expensive equipment

• Equipment is hard to use

• Varying reproducibility

• Protein denaturation or aggregation

Traditionally, physical techniques are used to disrupt cells, but there are disadvantages, such as?

1. Tissue disruption

2. Cell lysis

What are the first phases in cell fractionation?

To disaggregate the cells and break them open with minimal damage to the cellular fraction of interest

The goal of cell and tissue lysis

Properties of the source of sample

Choosing the best protein isolation method depends on the?

• Physical methods

  • Heat treatment

  • Homogenization

  • Sonication

• Chemical / Reagent-based methods

  • Treatment with detergent solution

Cell lysis have different methods. Cite examples.

Mechanical methods

It depends on the use of rotating blades to grind and disperse substantial amounts of complex tissue.

• Waring blender

• Polytron

What are commonly used for mechanical methods?

Waring blender

It is similar to the standard household blender

Polytron

It draws tissue into a long shaft containing rotating blades.

False.

In Polytron, the shafts vary in size to accommodate a wide range of volumes and can be used with samples as small as 1mL.

True or False.

In Polytron, the shafts vary in size to accommodate a wide range of volumes and can be used with samples as small as 1mm.

Liquid homogenization

It is the most commonly used technique for cell disruption.

Liquid homogenization

In this technique, cells are lysed by forcing the cell or tissue suspension through a narrow space, which is capable of shearing the cell membranes.

1. Dounce homogenizer

2. Potter-Elvehjem Homogenizer

3. French press

3 different types of homogenizers

Dounce homogenizer

It consists of a round glass pestle that is manually driven into a glass tube.

Potter-Elvehjem homogenizer

It consists of manually or chemical driven polytetrafluoroethylene pestle shaped to fit a rounded or conical vessel.

French press

It consists of a piston that is used to apply high pressure to a sample volume of 40-250 mL, forcing it through a tiny hole in the press.

40 - 250 mL

The sample volume in French press

French Press

The equipment is expensive, but the ___ is often the method of choice for breaking the bacterial cells mechanically

Freeze-thaw method

It is commonly used to lyse bacterial and mammalian cells.

• Dry ice / ethanol bath

• Room temp / 37 C

In Freeze-thaw method, the cell suspension is frozen in a ___/___ or freezer and then immediately followed by thawing at ___ temperature or ___.

Freeze-thaw method

This technique causes the swelling of the cells until they break as ice crystals.

The freeze-thawing process is repeated several times → lengthy

Disadvantage of freeze-thaw method

• Recombinant proteins

• Lysis

Although lengthy, freeze/thaw has been shown to release ___ ___ located in the cytoplasm of bacteria excellently and is recommended for the ___ of mammalian cells in some protocols.

Manual grinding

It is the most basic and common method used to disrupt cells.

• Liquid nitrogen

• Mortar and pestle

Manual grinding

First, the tissue needs to be frozen in ___ and then, crushed using a ___.

• Tensile strength

• Plant cells

• Manual grinding

Because of the ___ of the cellulose and other polysaccharides comprising the cell wall, for instance of ___, ___ method is the fastest and most efficient way to access plant proteins and DNA.

Sonication

This method uses pulsed, high-frequency sound waves to agitate and lyse cells, bacteria, spores, and finally diced tissues.

• Probes

• Vapor bubbles

• Shock waves

Sonication

Mechanical energy released from the ___ initiates the formation of microscopic ____ causing ____ to radiate through the sample.

Volumes

Sonication is best suited for?

The type of cell used

The frequency depends on?

Microscale cell disruption

It is often accomplished through chemical and enzymatic methods or combinations of the two.

• Rapid, gentle, efficient, and reproducible → high protein yield

• Extracts total protein or subcellular fractions

• Disrupts lipid membrane and cell wall

• Components are removed for downstream analysis

• High conc. of salt and detergents incompatible with protein assays and mass spectrometry

The characteristics of reagent-based methods for lysing cells

Lysozyme (200 ug/mL)

It can be added to digest polysaccharide components of yeast and bacterial cell walls, making the cells easier to lyse.

Glass beads

An alternative to lysozymes wherein the use of this method will facilitate the crushing of cell walls. This edition is commonly used with yeast cells.

• Increases

• DNase

• RNase

Glass Beads

The viscosity of a sample typically ___ during lysis due to the release of material that can be removed by adding ___ and ___ to avoid interfering with the protein target.

Protease inhibitors

However, in all of this, proteolysis can be a problem whenever cells are manipulated; this can be avoided by adding ____ to all samples due for lysis.

False.

Unlike nucleic acids, proteins do not have a generalized purification method.

True or False.

Unlike nucleic acids, proteins have a generalized purification method

• Chromatography

• Electrophoresis

Purification techniques of proteins wherein they are used to separate proteins into different bands depending on whether the goal is for preparatory step or for quantitative analysis.

• Liquid Chromatography

• Fast protein LC

• High-Performance LC

The types of chromatography that can be used for protein purification, depending on the goal (preparatory or quantitative analysis).

• Hydrophobic interaction column chromatography

• Size-exclusion chromatography

• Affinity chromatography

It is possible to use the following techniques either as a single step or a sequential connected step:

Column chromatography

It is a primary protein purification method in use in most laboratories.

• Cylindrical column → porous solid matrix

• Solvent → pumped

• Matrix → 2

Column Chromatography

• A combination of proteins in a solution is added to the surface of a ___ loaded with a ___ submerged in a solvent.

• After that, a large amount of ___ through the column is ___ through the column.

• Due to their association with the ___ , separated proteins are deluded ___ distinct levels, and they can be obtained individually as they flow from the bottom.

• Charge

• Hydrophobicity

• Size

• Ability to attach to specific chemical components (depending on matrix selection)

In column chromatography, proteins are separated according to?

Ion-exchange chromatography

Columns of ion exchange are filled with tiny beads holding either positive or negative charges that retard proteins of the opposite charge.

• pH

• Ionic strength

In Ion-Exchange Chromatography, the interaction of protein and matrix relies on the ___ and ___ of the fluid moving down the column.

• Binds to negatively charged beads

• Flows through

Ion-exchange chromatography

• Positively-charged protein

• Negatively charged protein

Gel-filtration chromatography

It separates proteins by size.

• Postponed and moved through the column

• Wiped out of the column

In gel filtration filtration:

• Protein molecules tiny enough to reach the bead

• Proteins that cannot enter the beads

Affinity chromatography

It is contained in a matrix covalently pair with a molecule that interacts specifically with the protein of interest.

• pH shift

• Concentrated salt solutions

Affinity chromatography

Proteins that attach specifically to such a column can eventually be produced through a ___ or ____, and these are extremely purified.

• Attaches → glucose residues

• + Glucose → Released

In affinity chromatography, the glucose-binding protein attaches to ___ residues on beads. Upon addition of glucose, the glucose-binding proteins are ___.

Colum Chromatography

What protein separation by chromatography?

Ion-Exchange Chromatography

What protein separation by chromatography?

Gel-filtration Chromatography

What protein separation by chromatography?

Affinity Chromatography

What protein separation by chromatography?

Western blot

It is used to detect antibodies to specific epitopes of antigen subspecies and confirm the specificity of antibodies detected by screening tests like ELISA.

Immunoblot

Other term for Western blot

• Electrophoretically

• Molecular weight

• Membranes

• Antibodies

In the Western blot technique, proteins are separated ___ according to their ___, transferred to ___, and identified through labeled ___.

Enzyme Immunoassays

They are similar to Western blot in a way where they use antibodies to detect antigens, but they are detected in microtiter plates (in vivo) than absorbent membranes.

• Sensitivity

• Specificity

• But cannot be quantitative

Lateral flow techniques provide?

1. Sample preparation

2. Electrophoresis

3. Transfer

4. Blocking

5. Antibody incubation

6. Detection

Steps in immunoblotting

Linear state

In sample preparation, the proteins from fold state will turn into?

Electrophoresis

Migration of proteins based o their molecular weight

Membrane

In transfer, the proteins travel from gel to?

Blocking

It is done to prevent nonspecific binding of antibodies, and to cover the empty spaces of the membrane.

Antibody incubation

Primary and Secondary antibodies are added to the membrane

Detection

The membrane is treated with a substrate specific to the enzyme linked to the secondary antibody.

• Loading buffer

• Linearized protein → negative charge → size

Sample Preparation

A ____ is added to the sample to separate the macromolecules in the sample. This will result in ___ protein with a ___ charge proportional to their ___.