CMB Cell Probing Tools

Light Microscopy

• visible range 0.4 - 0.7 micrometer (0.5 bacteria)

• limitations: molecules of low abundance in the cell cant be seen

Resolution and Wavelength

• resolution limit dependent on which wavelength of light is used

• smaller wavelength of light can enable visibility of small objects

• resolution = ~0.5(wavelength)

alpha (light microscopy)

angular aperture (half angle) of the cone of light entering the objective lens

N (light microscopy)

refractive index of the medium between the specimen and the objective lens

differential staining for light microscopy

malachite green, sudan black, coomassie blue, eosin

eosin

stain for light microscopy, which binds to the acidic cytoplasmic contents, including RNA and side chains of acidic amino acids

hematoxylin

binds Arginine rich nucleoprotein complexes and basic proteins

• mostly stains nucleus blue

Fluorescent Microscopy

• purpose: used for detection of specific proteins or other molecules

• fluorescent dyes covalently bind to any antibody (incubate sample with primary antibody, wash away unbound, incubate with fluorochrome conjugated secondary antibody, observe)

• resolution = ~20nm

• two filters used, one which allows only light of a specific wavelength

Fluorescent Proteins

• GFP (beta can structure)

• can be helpful in studies and for diagnostic purposes

Phase Contrast Microscopy

• resolution affected by interference whose amplitude is reduced

• examination of live cells and their movements

• exploits the difference in the phase of light passing through different regions of the specimen

differential interference contrast microscopy

• technique that introduces contrast to images of specimens which have little or no contrast when viewed using brightfield microscopy, have a pseudo 3D-effect

confocal scanning microscopy

• light/lase used for making virtual slices to create a 3D image (computer generated)

• for specimens which cannot be sliced into sections

electron microscopy (em)

• resolution is limited by wavelength of light

• use electrons due to their much shorter wavelength, allowing for significantly higher resolution imaging

transmission electron microscopy (tem)

• A high-energy electron beam passes through a thin sample.

• The transmitted electrons are focused to form an image with atomic-level resolution

scanning electron microscopy (sem)

• A focused electron beam scans the surface of the sample.

  • dependent on surface angles (texture)

• Secondary or backscattered electrons are detected to produce detailed 3D surface images

• specimen is fixed (freeze fractured), dried and coated with thin layer of heavy metal

  • creates a metal replica which is what’s used in the SEM

• used to study surfaces of whole cells and tissues

biochemical analysis limitations

• source

• quantity

• purity

use of protease in cell sorting

dissolves cell-cell connection and connective tissueu

use of EDTA in cell sorting

weakens cell-cell adhesion and prevents aggregation
(binds and traps cations which are essential for cell-cell adhesion)

centrifugation

separates bio entities by size and density utilizing different speeds

cell sorter

• specific cell types (COI) labeled with antibodies conjugated to fluorescent dye get negatively charged

• charged cells get deflected by a strong electric field into an appropriate container

• can select 1/1000 cells at 5000 cells per second

• keeps cells alive

cell culture

• observe and analyze growing cells

  • provide solid surface for growth as most tissue cells are not adapted to living in suspension

primary cultures

cultures prepared directly from tissue

secondary cultures

sub-cultured primary cultures

cell line

a population of cultured cells

contact inhibition

point where there is no more room for cells to grow

transformation

loss of contact inhibition, growth on top of one another, uncontrolled proliferation

immortal cell line

indefinitely propagated but still grow monolayer

transformed cell lines

• population of cells that have undergone genetic or epigenetic changes, allowing them to bypass normal growth controls, including contact inhibition

• obtained by tumor inducing virus/chemicals

• can be stored in liquid N indefinitely

organoids

• rudimentary organ grown outside of the body

• intestinal adult stem cells cultured in 3D matrix can form intestinal organoids

• so far developed include

  • brain, intestine, stomach, kidney, heart, tongue

• limited to development seen in fetus but not to adult

• can be helpful in testing drugs

cell fractionation

cells can be fractionated into its functioning organelles and macromolecules

broken cells aren’t alive but can/should maintain biological activity

microsomes

derived from ER following cell disruption, bc ER is so membranous, when the cell breaks so will the ER

cell disruption

can be due to ultrasonic vibration, osmotic shock or grinding and leads to cell homogenate

differential centrifugation

• cell homogenate processed by successive ultracentrifugation’s at different speeds

• cell components are separated based on size and density

• each fraction can be further purified by repeated centrifugations

rate zonal centrifugation

• separates things further than that in differential centrifugation

• by virtue of a difference in their sedimentation rates. The rate at which the particles sediment depends on their size, shape, and density and also depends on the density and viscosity at each point in the gradient.

cell free systems

• fractionated cell homogenates that maintain a biological function

• determination of the function of different cell components

• allows study of biological process free from other side reactions

purification of proteins

• purpose: to isolate one protein from a mixture of proteins (ideally biologically active) in preparative mode

biological assay

purpose: to uniquely identify the protein of interest in the collected fractions during purifications

(detection does not mean isolated/pruified)

Bio Assay TBP

will bind to radiolabeled double stranded DNA containing TATAWAW sequence

Bio Assay Catalase

may be identified by its ability to produce fructose from sucrose

Bio Assay FP

shining a UV lamp on tested

Analytical Purification Technique

• informative and diagnostic

• likely not provide protein in biologically active state or sufficient quantities for further quantities

Preparative Purification Techniques

• informative and provide biologically active molecules and sufficient quantities for further experiments

Partition Chromatography

• analytical techniques

• used to separate small molecules

• principle: separation is based on relative solubility of the sample molecules in the two solvents

• includes an absorbent material, solvent which absorbs more acts as stationary phase

Gel Filtration

separation based on size

(larger proteins move rapidly, smaller proteins move slowly)

Anion Exchange Chromatography

• beads in ion exchange are positively charged and will bind to negatively charged proteins

• proteins are fractionated according to their net charge

Cation Exchange Chromatography

• beads are negatively charged and will bind to positively charged proteins

cation

positively charged

anion

negatively charged

Hydrophobic Interactions Chromotography

• separation of proteins with exposed hydrophobic side chains

• principle: hydrophobic regions of molecules interact with a hydrophobic stationary phase under high-salt conditions

Affinity Chromotography

• takes advantage of biologically important binding interactions that occur on protein surfaces

• highly efficient

  • ex. matrix linked to enzyme to purify its substrate

high performance liquid chromatography

advanced form of liquid chromatography used to separate, identify, and quantify compounds in a mixture

• requires elaborate system of pumps and valves to force he solvent through the columns at sufficient pressure

SDS-PAGE

analytical

• Sodium dodecyl sulfate which is negatively charged binds in high numbers to hydrophobic regions of proteins causing unfolding and enforcing negative charge on protein

• rate of migration in electric field based on net charge, size and shape of molecule

(not sufficient enough to validate the purity of protein, can separate but not identify)

inert matrix

highly cross-linked gel of polyacrylamide

mercaptoethanol treatment

breakage of S-S bonds to release sub-unit polypeptides

Heat Treatment

denatures

2D Electrophoresis

step 1: separation based on charge by isoelectric focusing (determining molecule isoelectric pH)

step 2: sds-page at right angle to separate by size

isoelectric point

pH where molecule has 0 net electric charge
those with high isopH are more negative

those with low isopH are more positive

Southern Blot

DNA

detect specific DNA sequences within a complex mixture of DNA

DNA is separated by size and then denatured and single stranded DNA is obtained

They are then transferred to a membrane

A labeled DNA or RNA probe (a short sequence of nucleotides that is complementary to the target DNA sequence of interest) is applied to the membrane and binds to the complementary DNA sequence on the membrane.

The probe is typically labeled to allow detection.

Northern Blot

RNA

used to study gene expression

detects changes in relative abundance of selected RNAs detected by specific complementary nucleic acid probes

Western Blot

to detect and analyze SPECIFICALLY proteins. Identifies proteins in a complex mixture, determines size, and quantifying their expression levels
1. run through sds-page and then transferred to a membrane which maintains the pattern of the gel. The membrane is incubated with a primary antibody specific to the POI and is then incubated with a secondary antibody that is specific to the primary antibody which is usually labeled for detection

genomic library

made from genomic DNA and may represent the entire genome

multiple cloning site

region into which exogenous DNA can be inserted

plasmid vector

DNA cloning in a plasmid vector allows amplification of a DNA fragments

cDNA libraries

made from reverse transcription of total mRNAs produced by specific cell types in specific conditions

• contains representative copies of cellular mRNA sequences

• can be screened w radiolabeled probe to ID a clone of interest
  (cDNA is methylated to be protected)

expression library

cDNA libs made in expression vectors (need assistance from the host cell to express cloned cDNAs into their corresponding mRNAs and proteins)

• can generate biological active protein clones

x-ray diffraction analysis

used to determine 3D folded structure of a protein in crystallized form

NMR spectroscopy

can give distance between the parts of protein molecules

3d strucutre can be computed

limit: 15 to 20 kD

useful for functional domains

cryo-electron microscopy (CEM)

uses fast moving election beam to study biomolecular structures at atomic scale

can be used to visualize molecular movement, function and interactions

combat nearly all limits of NMR and XRD