MODULE 4: BIOPHYSICAL TECHNIQUES Flashcards
1
Q
Biophysical techniques provide information about
A
electrical structure
size
shape
dynamics
polarity
modes of interaction
2
Q
Methods used for gaining information about biological systems
A
Biophysical techniques
3
Q
Biophysical techniques study the
A
structure
properties
dynamics
function
4
Q
TRUE OR FALSE:
Biophysical techniques involve the development of novel techniques to investigate the structure, properties, and biological functions of biomolecules or the application of these techniques to monitor how the structure and dynamics of biomolecules enables specific biological functions
A
TRUE
5
Q
purify or isolate biological specimens (organisms, cells, and molecules) / get them ready for use in some other process or further experimentation
A
PREPARATIVE TECHNIQUES
6
Q
used to measure physical aspects (energy, pressure, volume, length, stress) of a biological system
A
ANALYTICAL TECHNIQUES
7
Q
machine used to spin a sample of material around in circles
A
centrifuge
8
Q
TRUE OR FALSE:
In ultracentrifuge, the force is similar to, but typically much smaller than, the normal force of gravity.
A
FALSE, much larger
9
Q
A centrifuge designed to speed an extremely high rate of speed
A
Ultracentrifuge
can exert forces as much as 1 million times that of gravity
10
Q
describes motion of particles in fluid under the application of a force
A
SEDIMENTATION
11
Q
sedimentation rate of a particle depends on
A
force
density of the fluid
size
density of particles
12
Q
what increases the sedimentation rate?
A
Applying force stronger than gravity
13
Q
convenient technique for separating molecules of different sizes
A
ULTRACENTRIFUGATION
USED AS BOTH PREPARATIVE AND ANALYTICAL
14
Q
Commonly used to isolate samples of pure DNA
A
ULTRACENTRIFUGATION
15
Q
Exosomes isolation
A
ULTRACENTRIFUGATION
16
Q
vesicles or cellular components that exist outside the cell
A
Exosomes
17
Q
contains special optical devices and sensors that can track the movement of molecules as they are being centrifuges
A
ANALYTICAL CENTRIFUGE
18
Q
used to detect conformational transitions and to determine the number of subunits making up molecular complex
A
Analytical Ultracentrifuge
19
Q
Can calculate the size and approximate shape of molecules
A
Sedimentation
20
Q
laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge
A
ELECTROPHORESIS
21
Q
It applies electric current to biological molecules
A
Electrophoresis
22
Q
used to move molecules to be separated through gel
A
electric current
23
Q
TRUE OR FALSE
Electrophoresis relies on sedimentation.
A
TRUE
24
Q
TRUE OR FALSE
In electrophoresis, the force results from an electric field applied to electrically charged particles or molecules.
A
TRUE
25
Common type of electrophoresis
gel electrophoresis
26
fluid that has a molecular structure that gives it properties similar to a solid
gel
27
# TRUE OR FALSE
Larger molecules are more easily obstructed than smaller molecules.
TRUE
| GEL INCREASES DIFFERENCES IN SEDIMENTATION
28
What affects the sedimentation in gel electrophoresis?
density of gel
size and shape of molecules
charge on molecules
| Molecules with more charge = stronger force generated by electric field
29
enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis
CHROMATOGRAPHY
30
coined the term chromatography in 1906
Russian Botanist Mikhail Tswett
31
Who described the first analytical use of chromatography in gas for the analysis of fatty acid mixtures?
James and Martin in 1952
32
Chromatographic procedures involve
size
binding affinities
charge
33
powerful separation tool used in all branches of science, used to separate components from complex mixtures
CHROMATOGRAPHY
34
factors effective on Chromatography
adsorption (liquid-solid)
partition (solid-liquid)
affinity
35
Because of the differences in separation caused by chromatography, some components of the mixture stay longer in what phase?
stationary phase
| move slowly in th system; mobile phase= move rapidly; leaves faster
36
Three components form the basIs of the chromatography technique
| BASIC COMPONENT EFFECTIVE ON THE SEPARATION OF MOLECULES FROM EACH OTHER
STATIONARY PHASE
MOBILE PHASE
SEPARATED MOLECULES
37
composed of a solid phase or a layer of a liquid adsorbed on the surface solid support
STATIONARY PHASE
38
always composed of liquid or a gaseous component
MOBILE PHASE
39
Four separation techniques based on molecular characterisctics and interaction type
Ion exchange
Surface Adsorption
Partition
Size Exclusion
40
chromatography techniques based on the stationary bed
column
thin layer
paper chromatography
41
# APPLICATION OF CHROMATOGRAPHY
- identify and analyze samples for the presence of trace elements or chemicals
- separation of compounds based on their molecular weight and element composition
- detects the unknown compounds and purity of mixture
- drug development
PHARMACEUTICAL SECTOR
42
# APPLICATION OF CHROMATOGRAPHY
- testing water samples and also checks air quality
- HPLC and GC are very much used for detecting various contaminants such as polychlorinated biphenyl (PCBs) in pesticides and oils
- life sciences applications
CHEMICAL INDUSTRY
43
# APPLICATION OF CHROMATOGRAPHY
- food spoilage and additive detection
- determining the nutritional quality if food
FOOD INDUSTRY
44
# APPLICATION OF CHROMATOGRAPHY
- analyzing blood and hair samples of crime place
FORENSIC SCIENCE
45
Used in protein separation like insulin purification, plasma fractionism, and enzyme purification
HPLC
46
Routinely practiced in the biopharmaceutical industry, along with the vaccine development, and gene therapy vectors
Virus purification
47
# APPLICATION OF CHROMATOGRAPHY
- HPLC
- Virus purification
Molecular Biology Studies
48
type of partition chromatography applied to isolate molecules with different molecular sizes
Size Exclusion Chromatography
49
it uses gravity and pressure to sediment a solution through gel
Size Exclusion Chromatography
50
tightly packed suspension of gel beads
SEC gels
51
# TRUE OR FALSE
In SEC, it is the larger molecules that pass through the gel faster and the smaller molecules lag behind
TRUE
52
Using gels with different size pores can --------------------
different size molecules from the insides of the gel particles.
exclude
53
study of the interaction between radiation and matter
spectroscopy
54
measure the intensity of the emergent radiation
spectroscopy
55
comon properties of spectroscopy
direction of the emitted radiation
polarity
56
resulting plot that has the function of the wavelength or the frequency of radiation
SPECTRUM
57
Formula for frequency
*f* = c/λ
| c = speed of light
λ = wavelength
58
Formula for Energy
E = h*f*
| h = Planck's constant: 6.626068 x 10^(-34) kg m^2/s or J/s
59
techniques of spectroscopy were developed using
visible light (380 to 750 nm)
60
spectroscopic techniques are classified according to
* type of light used (EM)
* properties of the emergent light measured
61
measure the amount of light absorbed as we control and slowly vary the temperature of a sample
temperature absorbance spectroscopy
62
Techniques that do not involve EM Rad
* Electron Spectroscopy
* Mass spectroscopy
63
measures the kinetoc energy of electrons that emerge from the sample
Electron Spectroscopy
64
produces a spectrum as a function of a mass
Mass spectroscopy
65
provide information about the identity of biological molecules, their structure, conformational transitions, binding and kinetics
Spectroscopic techniques
66
frequencies and amplitudes are perceive as -------------- respectively
color and intensity/brightness
67
measurement of the interaction between radiation and matter
Spectrometry
68
instrument which performs the measurements
Spectrometer or Spectrograph
69
# TRUE OR FALSE
EM is treated as photons or particles and waves
TRUE
70
Exhibit elastic collisions, bouncing and scattering off of objects
Photons
71
Exhibits reflection, refraction, and interference
Waves
72
also called absorbance spectroscopy
ABSORPTION SPECTROSCOPY
73
light of a specific wavelength is incident through a sample and measure the intesnity of the light that comes out the other side
ABSORPTION SPECTROSCOPY
74
In absorption spectroscopy, light is converted into
kinetic energy; higher or excited energy state
75
the absorbance of a given sample depends on three things
1. **intrinsic ability** of molecules in solution to **absorb light**
2. **concentration** of the molecules in solution
3. **path length** of the light
76
measure that accounts for both concentration and thickness
molar extinction coefficient
77
it is used to determine the proportion of hemoglobin with oxygen bound to it
absorbance spectroscopy
78
graph of the absorbance versus the wavelength or frequency of light
absorption spectrum
79
* used to identify types of molecules in a sample
* used to measure concentration of molecules in solution (once the identity of the molecule is known)
absorption spectrum
80
used to folloe conformational transitions and ligand binding
absorption spectroscopy
81
measures absorbance across a range of temperatures; useful for studying temperature-induced conformational transitions
temperature scanning absorption spectroscopy
82
electrons drop down from their excited state, emitting light in the process
FLUORESCENCE
83
caused by absorption
Fluorescence
84
# TRUE OR FALSE
Emitted light is longer than the absorbed light.
TRUE
85
Can be used to characterize molecules and to measure and follow conformational transitions and ligand binding
FLUORESCENCE SPECTROSCOPY
86
a small molecule or the specific part of a molecule that is responsible for the fluorescence
fluorophore
87
technique in which a fluorophore is attached to another molecule in order to track that molecule through some biological process
FLUORESCENT TAGGING
88
used to determine the sequence of residues in DNA
FLUORESCENT TAGGING
89
uses genetic tag that produces a stoichiometric ratio of a fluorescent protein reporter and the protein of interest during protein translation
PROTEIN QUANTITATION RATIOING (PQR)
90
Distinct Characteristics of NMR
* involves interaction of light (EM) with the nuclei of atoms in a molecule
* provide structural details (higher resolution)
* strong magnetic field that alters and limits some of the energy states available to the nuclei
* uses RF
91
Absorption and Fluorescent spectroscopy rely on EM in
UV
Visible
Infrared
92
# TRUE OR FALSE
The frequency of the EM radiation will be proportional to the energy difference between the two spin states.
true
93
# TRUE OR FALSE
the **smaller energy difference** between spin states means that these nuclei will resonate with **lower frequencies (less energy)** of EM
TRUE
94
# TRUE OR FALSE
Nuclei that are **less shielded** by electrons will be more exposed to the magnetic field; thus, these nuclei resonate with **higher-frequency** EM
TRUE
95
Molecules of parts of molecules are ionized and then passed through a magnetic field
Mass Spectroscopy
96
used both for determining molecular weights and identifying molecules (once the molecular weight is known)
MASS SPECTROSCOPY
97
technique for determining the relative positions of atoms within a crystal
X-RAY CRYSTALLOGRAPHY
98
orderly, three-dimensional, repeating arrangement of atoms or molecules
crystal
99
provides precise, high resolution structural information for molecules in a crystal
X-RAY CRYSTALLOGRAPHY
100
used to discover that DNA is a double helix
X-ray Crystallography
101
disadvantage of X-ray crystallography
molecules must be in crystalline form
102
occurs when light waves pass through an ordered arrangement of openings and interfere with each other on the other side
DIFFRACTION
103
Waves meet in phase with one another, the peaks from one wave come together with peaks from the other wave
| peaks become higherand troughs become lower
CONSTRUCTIVE INTERFERENCE
104
Meet out of phase, a peak and trough come together
| waves flatten out and lower the intensity of radiation
DESTRUCTIVE INTERFERENCE
105
Technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye
Microscopy
106
Who built the first electron microscope?
Ruska and Max Knoll
107
use a beam of highly accelerated electrond
ELECTRON MICROSCOPES
108
the beam passes through a very thinly sliced sample to provide an image on the other side; the image is made by focusing the electron beam onto a view screen coated with some material that fluoresces in response to the incoming electrons
TRANSMISSION ELECTRON MICROSCOPY
109
gradually scanned across the surface of the specimen
| some of the intensity of the elctron beam is lost
SCANNING ELECTRON MICROSCOPY
| 1/10 as powerful as TEM,but provides 3-dimensional image of the specimen
110
provides magnified images similar to those of SEM, but with resolution similar to those of TEM (10 times better than SEM)
ATOMIC FORCE MICROSCOPY
111
It works by moving a mechanical probe across the surface of the object being scanned
| provides true, three-dimensional information about the object
ATOMIC FORCE MICROSCOPY
112
More advanced techniques of ------------------ and -----------------keep the probe a constant distance from the object's surface in order to avoid the possibility of the probe damaging or deforming the surface of the object being scanned
AFM
SPM
113
Instrument that uses focused laser beams to create piconewton size forces that can be used to hold and manipulate microscopic particles anywher from 0.1 nm (size of an atom) to 10000 nm (size of bacterium)
optical tweezers
114
the phenomenon of focused laser beams holding a single particle in place in three dimensions
OPTICAL TRAP
115
useful for investigationg the mechanics of and forces associated with molecular motors
OPTICAL TWEEZERS
116
Used for sorting and separating cells of various types or for measuring the forces necessary for bending or breaking a DNA molecule
OPTICAL TRAP
117
Measure and characterize electric currents in cells, particularly in excitable tissue cells such as neurons
VOLTAGE CLAMP
118
The basis of this technique is the ability to insert a very fine microelectrode into the cell, with another electrode in contact with the fluid around the outside of the cell.
VOLTAGE CLAMP
119
Voltage is clamped, or held constant, through feedback mechanisk
VOLTAGE CLAMP
120
value of voltage that is held constant
command voltage
121
allows to measure the cell's electric current under a variety of conditions; useful to discover and characterize volatge-gated ion channels which are proteins found in cell membranes
VOLTAGE CLAMP
122
Used to keep the current constant while allowing voltage to vary
CURRENT CLAMP
123
The electrode is placed inside a micropipette filled with an electrolyte solution, and the micropipette is placed against the cell membrane
| ALLOWS TO INVESTIGATE THE BEHAVIOR OF A SINGLE ION CHANNEL
PATCH CLAMP
124
SIMILARITIES OF VOLTAGE CLAMP, CURRENT CLAMP, AND PATCH CLAMP
they all consist of an electrode, some wires, and electronic feedback unit capable of measuring and manipulating volatges and currents
125
difference among current clamp, voltage clamp, and patch clamp
settings on the device:
MEASURE CURRENT OR VOLTAGE
HOLD FIXED
MANIPULATE VIA FEEDBACK MECHANISM
126
Measure of energy changes in the form of heat
CALORIMETRY
127
instrument that measures heat energy
CALORIMETER
128
used to measure the amount of energy absorbed or released
CALORIMETRY
129
* Used to determine the amount of energy necessary to unwind a piece of DNA helix
* Used to measure the binding strength of various drugs to a particular protein
CALORIMETRY
130
useful for measuring the very small amounts of energy
MICROCALORIMETERS
