Lecture 2 Spectrophotometry, Assays, Media Growth Flashcards
Characteristics of an Assay
- Physical property that can be observed and measured (ex. colour, radioactive emission, sound)
- Sensitive to changes in concentration
- Sensitive to a specific substance
- Changes are predictable and quantifiable
- Capable of calibration
Principles of spectrophotometry
Biological chemicals absorb light in the UV-Vis range due to resonance of bonds
Need relationship between given compound and its absorption characteristics to detect and measure that compound in a given solution
Direct or indirect spectrophotometry
What wavelength does DNA and RNA (nucleic acids) absorbs at?
Around 260 nm
What is lambert’s law?
A = log(1/T) = K1
For each unit length of light path (l), transmitted light is reduced by constant proportion (20%)
As absorbance (A) increase, transmittance (T) decreases
What is Beer’s Law?
Beer Law: K = ε[c]
What are the differences between Lambert’s Law and Beer’s Law?
Lambert’s Law: Absorbance is inversely proportional to transmittance
Beer’s Law: Absorbance is proportional to concentration and path length (with extinction coefficient)
What is Beer-Lambert Law?
Absorbance is directly proportional to solute concentration and to the light path length
A = Kl = εcl
As ε increases, absorbance increases
As concentration increases, absorbance increases
Beer-Lambert Law Variables
ε - extinction (absorption) coefficient is related to electronic environment of light-absorbing unit
Anything that affects electronic environment, changes ε: pH, ionic strength, solvent
Defined for a particular solvent at a particular wavelength (usually independent of concentration)
A - absorbance of the sample -> generally written as OD (optical density)
C - concentration
l - path length (1 cm)
What is a spectrophotometer and how does it work?
- Separates light into different wavelengths
- Detects “I” and amplifies signal (Io is incident light and I is transmitted light)
- Signal is converted to read-out
The wavelength that is transmitted is the wavelength that can not be absorbed by the specimen
Limitations of spectrophotometer
Once the absorbance is above 1.0, it is not as accurate of a reading since almost all light is absorbed
A reliable range is between 0.05 - 1.2
What is Direct spectrophotometry and what are the set values?
Measures values directly without using secondary reagents (only requires compound of interest) using physical characteristics of the material being tested
For pure nucleic acid:
1 OD260 = 50 ug/mL of DNA
1 OD260 = 40 ug/mL of RNA
1 OD260 = 33 ug/mL of SS-DNA
Examples
0.5 A = 25 ug/mL of DNA
0.5 A = 20 ug/mL of RNA
What wavelength does amino acids absorb at?
Aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe) at 280 nm
These AA have different extinction coefficients -> makes it difficult to relate absorbance to concentration
What are the concentrations of amino acids in direct spectrophotometry?
Not all proteins contain the same # of AA therefore there are no set concentration values
Approx. 1 OD280 = 0.4-1.5 mg/mL of protein
Rough rule: if sample containing pure protein has an absorbance of 1 at 280 nm, the it contains 1 mg/mL of protein
How does other wavelengths affect the concentration of protein for direct spectrophotometry?
Peptide bonds absorb 190-230 nm
OD205 = ~15 mg/mL
How to estimate the purity of a solution using UV direct spectrophotometry?
Ratio of two absorbance values at 260 nm and 280 nm (A260/A280)
Pure RNA = 2.0
Pure DNA = 1.8
Pure Protein = 0.6
A ratio of 1.8-2.0 is desired when purifying nucleic acids (can’t distinguish between DNA and RNA)
A ratio less than 1.7 means there is contaminants in the solution either protein or phenol
What is turbidity?
Turbidity can be referred to the cloudiness of solution
- Can cause apparent increase in absorbance of a sample
- Leading to incorrect readings
Background correction
- If a sample absorbs at 320 nm, the absorbance is due to turbidity
- Since proteins and nucleic acids do not absorb at 320 nm
- The absorbance at 320 nm can be subtracted from the readings at 260 nm and 280 nm
What is it called when 50% of the DNA is denatured at a certain temperature?
Tm or Melting temperature
What are the characteristics of indirect spectrophotometry?
- Compound of interest is colourless
- Can react compound with a secondary reagent to produce a colour product
- Measure Abs of coloured product -> directly related to [original compound]
- Colour reagent must be in excess for this to be true (all proteins will react and change colour)
- Compound of interest always consumed (sample cannot be reused)
What techniques are used for indirect spectrophotometry?
Determine the concentration of an unknown protein sample using a set of standards
Spectronic 20 (Spec 20) spectrophotometer - using glass cuvettes and only works in visible wavelengths
Types of indirect measurement of protein
- Bradford method
- Lowry
- Biuret
- BCA (Bicinchoninc Acid) assay
Bradford Assay/Dye Binding Method
- Coomassie Blue G-250 dye (Brown to blue)
- After reacting with protein, the dye shifts Amax from 465 nm (brown) to 595 nm (blue)
- The stronger the blue, the higher the protein concentration
- Inhibited by detergents
- Prepare standard curve and calculate [protein]
Limitations and Characteristics of Braford Assay
Detection limitations: 1-20 ug (micro assay) and 20-200 (macro assay)
Characteristics:
- Fast process and inexpensive (15 min - 60 mins)
- Very sensitive
- Compatible with a wide range of substances
- Dye reagent in complex is stable for approx. one hour
Considerations:
- Dye binds to quartz cuvettes, better to use glass or plastic ones
Preparation of Bacterial Media
- Media is the nutrient source to grow bacteria
- The media you would have made in the lab is called LB (Lysogeny broth) - a nutrient rich media at pH ~7
- Media is sterilized in an autoclave before use -> heats the media at high temperature (121C)
- If necessary can add an antibiotic to the media -> prevents unwanted bacteria from growing
- Pour plates: must wait for media to cool to around 50C
