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PROTEINS
Bio150 Lab
Prepared by: Jassy Mary S. Lazarte & Jeremy G. Vicencio Department of Biology, University of the Philippines Manila
Protein from the Greek proteios (primary, first, and foremost) Proteins are the work horses of biological systems.
They play key roles in constructing and maintaining living cells Our genes code for proteins Proteins are polymers of amino acids
Proteins are
Enzymes (biological catalysts) Hormones Storage proteins Transport proteins Structural proteins Protective proteins Contractile proteins Toxic proteins
*Upon lysis of the cell, proteases are released into the lysate
Protein Isolation
Protein Isolation
protein isolation is carried out at low temperature to minimize the activities of these protease to further optimize the results, use the proteases inhibitors
Protein Isolation
3. Addition of Protease Inhibitors EDTA (or EGTA): chelating the Ca2+ PMSF: a general serine protease inhibitor.
*It is the most common inhibitor used in protein purification. *Soluble in isopropanol.
4. Quantify Protein
Protein Quantitation
A. Colorimetric methods: 1. Biuret 2. Lowry 3. Bradford B. UV absorption method
Protein Quantitation
some proteins do not contain these amino acids, it will not absorb UV light Nucleic acids (DNA, RNA) contaminant will also absorb UV light
Protein Quantitation
% protein
% NA
100 95 90 70
0 5 10 30
100 95 90 70
0 5 10 30
Protein Quantitation
Colorimetric methods
protein
samples can be modified with appropriate reagents to produce a color reaction and measure protein concentration using a spectrophotometer.
BRADFORD ASSAY
Bradford Method A dye known as CBBG, Coomassie Brilliant Blue G-250 was developed by the textile industry. It was noticed to stain skin as well as the textiles. This dye (which normally absorbs at 465nm) binds to proteins and to absorb strongly at 595nm. The assay is sensitive, but somewhat non-linear
Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976. Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
Advantages
Fast
and inexpensive Highly specific for protein Very sensitive [1-20 g (micro assay) 20-200 g (macro assay)] Compatible with a wide range of substances Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin) Dye reagent is complex is stable for approximately one hour
Disadvantages
Non-linear
standard curve over wide ranges Response to different proteins can vary widely, choice of standard is very important
Absorption spectra of anionic and cationic forms of the dye overlap. So the standard curve is non-linear although all kit providers of the Bradford assay insist that the assay performs linearly. The assay performs linearly over short concentration stretches. If your sample is more than 20 micrograms, a second order curve will fit much better than a linear curve.
Points to remember:
What is the relationship of the absorbance reading with the protein concentration? Directly proportional What is the identity of the standards? bovine -globulins
Hydrophobic interaction - association of nonpolar molecules that minimizes exposure to the polar surroundings Chromatography - a technique used in which a mixture of dissolved components is fractionated as it moves through a certain type of porous matrix
HIC
Alternatives
Gel
Why HIC?
Different basis of separation Weaker interactions
Separation principles
HIC: Purpose
Downstream purification Separation of biomolecules Exploits differences in hydrophobicity. Number of hydrophobic amino acids.
HIC: Principle
Separation of substances is based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix. How is this achieved?
HIC: Principle
HIC: Principle
HIC: Applications
HIC has found many applications in the purification of a wide variety of proteins, including Monoclonal antibodies Vaccines Truncated forms of r-proteins Interferons EGF (epidermal growth factor) Human growth hormone
HIC: Advantages
Large volume of sample can be loaded Samples with high ionic strength can be used Well suited to use before gel filtration, ionexchange and affinity chromatography Sample eluted with low salt Purification steps that generate large sample volume can be coupled with this method
HIC: Advantages
Good for samples after ammonium sulfate fractionation. Sample can be used in ion exchange chromatography step
and concentration of ligand Type of base matrix Type and concentration of salt pH Temperature Additives
Buffers
Equilibration buffer, 2 M (NH4)2SO4- used to prime the column for the binding of proteins Binding buffer, 4 M (NH4)2SO4- hydrophobic region is more exposed causing it to interact and bind with the hydrophobic regions of the matrix Wash buffer, 1.3 M (NH4)2SO4-use - wash the weakly associated proteins from the column, while the strongly hydrophobic proteins are bound to the matrix Elution buffer, 10 mM Tris/EDTA, -wash the strongly hydrophobic proteins from the column
fraction: hydrophilic proteins 2nd fraction: the rest of hydrophilic plus some hydrophobic proteins 3rd fraction: strongly hydrophobic proteins
the
last fraction that has the highest absorbance reading contains the most hydrophobic proteins
ELISA
Enzyme-linked Immunosorbent Assay
What is an ELISA?
Solid
phase (sorbent)
Allows
one to wash away all the material that is not specifically captured
Enzymatic
Allows
amplification
you to turn a little capture into a visible color change that can be quantified using an absorbance plate reader
Measure antibody levels (allergies, vaccines) Detect viruses (hepatitis, HIV, venereal diseases) Detect hormonal changes (pregnancy) Detect circulatory inflammatory markers (cytokines)
Advantages
10 g/ml - 1 mg/ml
High molar extinction coefficient (i.e., strong color change) Strong binding between enzyme and substrate (low KM) Linear relationship between color intensity and [enzyme]
Antibodies
Specificity Diversity hypervariable region (2020 ~ 1026 combinations; humans make ~ 108) Affinity range 105 < K < 109 M-1
ELISA
Enzyme Linked Immunosorbent Assay (ELISA) Term Was Coined By Engvall and Pearlmann in 1971 Different Types Sandwich Indirect Competitive Similar To RIA(radio-immuno assay), Except No Radiolabel Can Be Used To Detect Both Antibody and Antigen Very Sensitive, pg/mL Relies on Monoclonal Abs
ELISA-Types
ELISA
REAGENTS: Ag: chicken -globulin 1Ab: rabbit anti-chicken polyclonal Ab 2Ab: goat Ab conjugated to horseradish peroxidase(HRP) -substrate: 3,3,5,5-tetrametylbenzidine(TMB)