Mirco Lab Midterm: Lecture 7 Flashcards
Quantitation of biomolecules after
separation by electrophoresis. ( Both In direct and Direct)
Indirect by densitometry: after light (image) from
chemiluminescent reactions is first captured on Xray film or after illumination of stained gels
Direct - measurement of the emitted light of
fluorescently labeled proteins or nucleic acids,
from the enzymatic chemiluminescent reactions
after Western or from storage phosphor screens
after exposure to radioactively-labeled
proteins/nucleic acids
Principles of densitometry
It measures optical density: intensity of staining of bands
in gel or the degree of darkness of X-ray film
- Measures the degree of darkness as a function of light
transmission. The optical density is the Log of reciprocal
of transmittance: - OD = Log10 (1/Transmittance) = Ecl
- Transmittance: fraction of light not absorbed by the
sample; E: absorption coefficient; c: concentration of the
absorbing molecules; l: light path length - It requires a reference standard curve for quantitation
Benefits of protein quantification after
electrophoresis
The method allows the evaluation of purity, yield or
percent recovery of individual proteins in complex
sample mixture
- Quantification is relative if one protein is quantified
from a standard curve made with another protein
(i.e. BSA) - Quantitation is absolute if the protein is quantified
using a calibration curve generated by a range of
known concentrations of the same protein
Define Standard curve
- Standard curve is created by plotting the OD of
series of protein standards with increasing
concentration run on electrophoresis and stained.
It is used to interpolate the unknown
concentration of the protein of interest. - Data for the standard curve can be fit to any model
– linear, hyperbola, quadratic or any polynomial.
The goal is that the curve is smooth and comes
close to the data (OD of the standards)
Describe the image captured on X-Ray film
Film contains light-sensitive silver halide crystals: silver grains
- Photons activate silver grains converting some silver ions into
silver atoms, creating “latent image” - During the film processing the silver atoms in each activated
silver grain catalyze the conversion of the entire grain into
black metallic silver creating a visible image on the film - Unexposed silver grains are dissolved and washed during
fixation - Darkening of the film after development as a result of metal
silver deposit is called optical density (OD)
Mechanism of silver grain activation
- The film response to light is governed by the reciprocity law:
- Light intensity X duration = total exposure
- When signals are very strong or very faint the film response is
no longer proportional to intensity and duration, which is
called reciprocity failure - The non-linear response of film to strong signals is due to
saturation - Since multiple photons must hit a silver grain to create latent
image, when signal is very faint a stable latent image is unlikely
to form, with less probability for metal silver to accumulate –
the film becomes less responsive, the sensitivity drops
What are Imaging systems (detection systems)?
There are many imaging systems designed to
capture signals from different techniques and
samples i.e. fluorescence, chemiluminescence,
visible light
- In all systems the energy of photons is converted
to electrical charge/voltage proportional to the
number of photons using either a photodetector
such as photomultiplier tube (PMT) or a detector
array such as charge-coupled device (CCD)
What is Digitalization?
After emitted light is detected, converted to charge (voltage)
and amplified (PMT) the analog (electrical) signal is
converted to a digital signal by an analog-to-digital convertor.
- Digitalization turns a measured continuous analog signal
into discrete numbers by introducing predetermined
intensity levels - The number of intensity levels determines the digital
resolution of the instrument and is indicated by the number
of bits. Instruments with higher digital resolution will be
able to resolve two signals with only slightly different
intensities
Acquisition of digital image
Light from the fluorescent label, chemiluminescence
reaction or collected after illuminating X-ray film
or stained gel is detected and quantitated with a
variety of scanners or digital imagers based on
PMT or CCD
- The image is stored as digital data by dividing it
into a grid of very small regions “pixels” - Pixels are characterized by size, coordinates
(location) on the image and intensity
Define Image Depth
- Each pixel has intensity value describing its brightness
- Discrete number (bits) of pixels intensity values determine digital
resolution of the instrument (number of bits, image depth, bit
depth) or how well two signals with close intensities can be
resolved - Bits describe the total number of gray shades, based on exponent
of 2, that the CCD chip can distinguish - With 8 bits each pixel can be represented in 256 (28
) gray shades;
16 bits: 65536 (216); human eye is 6 bits - A chip with more bits can distinguish smaller differences in
intensities of compared images (such imaging system has wider
dynamic range)
Define Dynamic/linear dynamic range
- Dynamic range: range of band intensities that a detection
system can measure in a single measurement. It is related to
the bit depth and depends on system noise (added signal,
which is not part of the signal from the imaged object) - Instruments with wide dynamic range have high sensitivity
(detection of faint bands) without saturation of detection
(of strong signal bands) - Linear dynamic range: Instrument measured signal
intensities that are linearly related to the amount of the
source of the signal (protein or nucleic acid band on the
membrane or in gel)
Compare the imaging systems
Systems for direct capture of light such as systems for
fluorescence imaging, chemiluminescence or
phosphor screens have broader dynamic range, wider
linear dynamic range and are better for quantitation
Measurement of optical density of X-ray film, which
has been exposed to light source or the optical density
of stained gels has narrower dynamic/linear range
because of the specific photochemistry of the film and
the spectral properties of the stains
Explain the CCD camera’s performance
The most appropriate choice of label or stain will enhance
the performance in terms of both sensitivity and dynamic
range. For example fluorescent labels and stains care
more sensitive and have broader dynamic range than
colorimetric stains.
- CCD arrays are sensitive to light, temperature, high energy
radiation. The noise caused by dark current from thermal
energy can have a strong effect on instrument
performance. Cooling the camera significantly reduces
the noise level improving the sensitivity and dynamic
range
Imaging chemiluminescent Western blots: CCD vs. scanner
- Limitations of the CCD systems: since CCD
chips are small (~1cm2
) and the blot much
larger, imaging optics and long optical path are
required to project the entire blot area onto
the chip. This decreases light collection
efficiency, therefore requiring long integration
times to collect sufficient signal. As a
consequence, CCD cameras must be cooled
(expensive!) to decrease the dark noise.
Digital blot scanner
- Maximizes light collection to increase
sensitivity: the sensors of the scanner are very
close to the blot (~1mm) increasing light
collection 100-400 fold compared to CCD
camera allowing comparable signal to be
collected in a very short time (exposure)
during the scanning. This produces less dark
noise and allows light collection at room
temperature with low price sensors
