[1] protein analysis
Protein Analysis
Proteins
most abundant macromolecule in biological system
polymers comprising amino acids that are linked by peptide bonds.
diverse in their chemical and physical properties
hydrophobic/hydrophilic
acidic/basic/neutral
difference in length
function
antibodies
enzymes
messengers
structural components
transport
transport or storage molecule
biomarkers
they are the proteins that can be detected in the blood of the patient
A measurable substance in the blood that indicates diseases or environmental exposure
MOLECULAR SEPARATION OF PROTEIN MOLECULES
COLUMN CHROMATOGRAPHY
separation of soluble components in a solution by specific differences in physical-chemical characteristics of the different constituents
Uses a glass or plastic tube filled with resin
Most common method → for purifying proteins
protein sample is applied to the top of the column
sample migrate through the column at different rates based on → nature on the matrix and the physical and chemical properties of the proteins
targeted protein can be separated from others
GEL/GEL PERMEATION/ GEL FILTRATION/SIZE EXCLUSION
column matrix also known as → resin
consists of microscopic beads of inert material.
The resin bead has many tiny pores like a → whiffle ball
Larger molecules = emerge first
smaller molecules = emerge last
separate proteins based on the size and shape of a gel filtration column
ION EXCHANGE CHROMATOGRAPHY
separates molecules based on positive and negative charge
positively charged beads = negatively charged proteins will bind to the column matrix at low salt
Proteins → induced to dissociate with high salt.
Uses a resin to separate proteins on surface charges.
contains a resin bearing of either positively or negatively charged chemical groups
positively charged groups attract negatively charged solutes and are referred to as → anion-exchange resins
in low-salt solution, negative proteins bind to → positively charged anion-exchange columns
bound proteins in high-salt solution can be eluted through → high-salt buffer
Resins with negatively charged groups are cation exchangers
AFFINITY CHROMATOGRAPHY
isolate molecules based on ligand binding abilities.
uses lock and key binding that is widely present in biological systems
principle = protein binds to a molecule for which it has a specific affinity
proteins carry biological activity through → binding or complex formation with specific small molecules/ ligands
Ligand
the specific affinity where the protein binds to carry out its biological activity
The protein of interest is removed from the mixture of samples
The protein is eluted from the resin by adding high concentrations of free ligands in the solution.
relies on the biological specificity of the protein of interest
very efficient procedure
separate and prepare larger quantities of proteins and antibodies for study
depends in
specificity of binding between the affinity reagent on the resin
the molecule to be purified
can be made to purify a protein in a single step.
HYDROPHOBIC INTERACTION CHROMATOGRAPHY
proteins passed through a chromatographic column packed with a support resin to which the hydrophobic groups are covalently linked
Phenyl Sepharose
which contains a phenyl group
commonly used in this method
protein sample is prepared in a high-salt buffer
Proteins can be differentially eluted using a low salt concentration buffer or by adding solvents such as polyethylene glycol to the elution fluid
MOLECULAR SEPARATION THROUGH ELECTROPHORESIS
ELECTROPHORESIS
different purposes
for separation
to determine the sizes, presence, or amount of DNA
For proteins, we use polyacrylamide gel electrophoresis (PAGE) with SDS.
carried out with a stacking gel and separating gel.
associated with staining method → can detect bands of protein in a simple and relatively rapid manner.
very first qualities that need to be assessed for any protein sample
integrity
purity
SODIUM DODECYL SULFATE (SDS-PAGE)
Anionic detergent (-)
Disrupt the structure of the protein to make it linear and binds most protein
a detergent that disrupts protein tertiary structure
Both heat and reducing agents that can also denature the protein and break the covalent bonds between subunit
DTT
2-mercaptoethanol
harmful and poisonous
avoid skin and eye contact
often used to determine the molecular weight of proteins
The hydrophobic tail of SDS interacts strongly with polypeptide chains
The number of SDS molecules bound by a polypeptide = length (the number of amino acid residues) of the polypeptide
contributes two negative charges
After reduction and denaturation, protein migrate in the gel according to their molecular mass, allowing to detection of potential contaminants, proteolysis events
The electrophoretic mobility of proteins upon SDS-PAGE is inversely proportional to the logarithm of the protein’s molecular weight
ADVANTAGES OF SDS
coats all the polypeptides with negative charges
Once it becomes linear, purely (-) charges will be left; hence, it will be towards the anode or (+) charge.
masks the natural charges of the subunits
electrophorese according to their molecular masses
Small polypeptides = migrate rapidly.
Large polypeptides = migrate more slowly.