Transcript Assaying

Course is an Introduction to Biotechnology with emphasis on lab
methods
Unit 1 Cells used and growth of cells
Unit 2- DNA and DNA techniques
Unit 3- Protein and Protein techniques
Why are these things important in biotechnology
-e.g. 1. To get a new protein product (a drug?) formed in cell have to be
able to
• grow cell,
• recognise contaminants,
• measure growth,
• isolate new DNA to insert and perhaps cut or amplify it,
• insert new DNA into cloning vector like a plasmid,
• transform a bacterial cell
• grow cell, monitor for protein production and then isolate protein
-e.g. 2 Use all techniques to ask questions about how cells work at a
molecular level
-e.g. 3. Use techniques to develop diagnostic techniques
Assaying Amino acids, and Proteins
1.Amino acids
Structural building blocks of proteins
20+ amino acids, each differing only in the composition of the R groups.
An R group could be a sulfydrl, another methyl, a string of methyls, rings of
carbons, and several other organic groups.
The general formula for an amino acid is represented in the following ways
Parts of the Amino Acid:
a) Amino group (NH2)
b) Carboxyl group (COOH)
c) R-group: variable-
2. Proteins
Amino acids linked together sometimes with a non amino acid component
Peptide is a short stretch (~50aa) of aa.
Peptide bonds: Bond formed when 2 amino acids bond.
peptide bond (See vertical arrow).
Dipeptide- 2 amino acids joined by peptide bond.
Polypeptide- many amino acids bonded together.
Primary structure: specific amino acid sequence
Secondary structure: H bonding to side groups, forms folds or coils
Tertiary structure: Three dimensional shape. Final folded shape driven by
hydrophobic interactions
Quaternary structure: two or more polypeptide subunits linked
Protein assays
Protein quantitation is often necessary before processing protein samples for
isolation, separation and analysis by chromatographic, electrophoretic and
immunochemical methods.
The criteria for choice of a protein assay are usually based on:
•
convenience,
•
availability of protein for assay,
•
presence or absence of interfering agents,
•
need for accuracy
Assays can be quantitative or qualitative
Need to be concerned with
•
Accuracy-proximity to true value
•
Precision- agreement between relicate data
•
Concentration range- range within which the method is accurate
•
Detection limit-minimum concentration that can be detected with a particular
confidence level
•
Selectivity- extent method is free from interference due to other substances
in the sample
•
Sensitivity- ability to discriminate between small differences in analyte
concentration
•
Validation- process where accuracy and precision are checked in relation to
specific standards
Assays
• Some labs have strict validation rules e.g.
forensic labs. These include
• adherence SOPs
• calibration of assays using certified
reference material
• effective quality assurance and control
systems
• detailed record keeping
Absorbance assays
Monitors the absorbance of aromatic amino acids, tyrosine and tryptophan or
if the wavelength is lowered, the absorbance of the peptide bond. Higher
order structure in the proteins will influence the absorption
Absorbance at 280 nm
Range: 20 micrograms to 3 mg
Accuracy: Fair
Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets
Advantages
Quick
Sample can be recovered
Useful for estimation of protein before using a more accurate method
Well suited for identifying protein in column fractions
Disadvantages
Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets
Highly susceptible to contamination by buffers, biological materials and salts
Protein amino acid composition is extremely important, thus the choice of a
standard is very difficult, especially for purified proteins
Absorbance is heavily influence by pH and ionic strength of the solution.
This is often used to estimate protein concentration prior to a more sensitive
method so the protein can be diluted to the correct range
Colorimetric assays
1. Modified Lowry
• The first step is a Biuret reaction which reduces
Cu+2 to Cu+1
• The second reaction uses Cu+1 to reduce the
Folin-Ciocalteu reagent (phosphomolybdate and
phosphotungstate). This is detectable in the
range of 500 to 750 nm
–
–
–
–
Range: 2 to 100 micrograms
Accuracy: Good
Convenience: Fair
Major interfering agents: Strong acids, ammonium
sulfate
Colorimetric assays: Modified Lowry
Advantages
• Sensitive over a wide range
• The most commonly referenced procedure for protein determination
• Can be performed at room temperature
• 10-20 times more sensitive than UV detection
• Can be performed in a microplate format
Disadvantages
• Many substances interfere with the assay
• Alkaline copper reagent is laborious to prepare and will develop carbonate
scales over storage which interfere with optical activity, thus it must be
prepared fresh daily
• Takes a considerable amount of time to perform
• The assay is photosensitive, so illumination during the assay must be kept
consistent for all samples
• Amount of color varies with different proteins
• Since reduced copper is detected in the procedure, make sure that the
distilled water used in the procedure is fed from plastic lines and not copper
lines
Colorimetric assays
2. . Bradford assay
– Range: 1 to 20 micrograms (micro assay); 20 to
200micrograms (macro assay)
– Volume: 1 ml (micro); 5.5 ml (macro)
– Accuracy: Good
– Convenience: Excellent
– Major interfering agents: None
• Absorbance shift in Coomassie Brilliant Blue G250 (CBBG) when bound to arginine and
aromatic residues
• The anionic (bound form) has absorbance
maximum at 595 nm whereas the cationic form
(unbound form) has and absorbance maximum
at 470 nm
• 1-20 µg (micro assay) 20-200 µg (macro assay)
Bradford assay cont.
Advantages
• Fast and inexpensive
• Highly specific for protein
• Very sensitive
• 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
• Absorbance spectra of the two Coomassie Brilliant Blue G-250 species
partially overlap making the standard curve very important
• Non-linear standard curve over wide ranges
• Response to different proteins can vary widely, choice of standard is very
important
• The dye binds to quartz cuvettes so it is usually better to use glass or plastic
cuvettes
• The dye reagent is usually more convenient to purchase than to make, due
to the use of phosphoric acid
Colorimetric assays
3. BCA (Bicinchoninic Acid )
• The first step is a Biuret reaction which reduces Cu+2 to Cu+1
• In the second step BCA forms a complex with Cu+1 which it purple colored and is
detectable at 562 nm
• Range:0.2-50 µg
• Accuracy: Good
• Convenience: Good
• Major interfering agents: Ammonium salts
•
•
•
•
•
•
•
Advantages
Less susceptible to interference from common buffer substances
Very sensitive and rapid if you use elevated temperatures
Compatible with many detergents
Working reagent is stable
Very little variation in response between different proteins
Broad linear working range
•
•
Disadvantages
The reaction does not go to completion when performed at room temperature or
37oC. This can be a problem if you are assaying a large number of proteins
Dilution is often necessary for concentrated protein samples
•
Assaying Enzymes
• When assaying enzymes want to know how
much active enzyme you have.
• 2 types of assay 1) fixed time assays, 2) kinetic
• Fixed time assays are based on a single
measurement and the relationship is known to
be linear
• Kinetic assays are based on product formation
per unit time e.g grams per minute. Linearity has
to be established.
• Enzyme activity measured in Units
Example
Lactate + NAD+
LDH
↔
Pyruvate + NADH + H+
LDH = enzyme lactate dehydrogenase
• The activity of LDH can be measured by measuring the rate of
formation of the product NADH which can be measured by a change
in absorbtion at 340nm.
• The rate of formation of the product is dependent on the
concentration of substrates and the concentration of products and
the activity of the enzyme.
• If start with lots of substrate and active enzyme the reaction will go
forward and produce products
• As the product concentration increases the forward reaction will slow
down
• Build up of product pushes the reaction backwards
• To get a good measure of the enzyme activity need to measure it in
the beginning of the reaction before the concentration of the
products builds up enough to have an influence.
• Before the concentration of products builds up the
relationship between enzyme activity and product formation
is linear. As the product accumulates this linear relationship
often breaks down.
• When you do an enzyme assay need to ensure it is in the
linear range so need to plot the measurement (absorbance
usually) versus time to see whether linear.
• Use only the linear range.for calculation of number of units
Enzyme activity is calculated by measuring it over short time periods to
determine the initial velocity. Data from the linear portion of the curve only
must be used
Units of activity are calculated as below:
Units =
change in absorb/change in time (min) x 10 6uM/M x volume
*6220 M -1 cm-1
= umol/min
*6220 M -1 cm-1 = extinction coefficient for NADH, used to convert absorbance
to moles. This would differ depending on the product.