Cellular and

Molecular Biology
Techniques

1
 Cell Culture
• Tissue Culture is the general term for the removal of
cells, tissues, or organs from an animal
• their subsequent placement into an artificial
environment - a suitable culture vessel containing a
liquid or semisolid medium - nutrients essential for
survival and growth.

2
 WORK AREA AND
EQUIPMENT
• Laminar flow hoods - continuous displacement of air that
passes through a HEPA (high efficiency particle) filter that
removes particulates from the air
• CO2 Incubators. an atmosphere of 5-10% CO2 because the
medium used is buffered with sodium bicarbonate/carbonic acid and
the pH must be strictly maintained. Culture flasks should have
loosened caps for gas exchange
• Microscopes. Inverted phase contrast microscopes
• Vessels. Anchorage dependent cells require a nontoxic,
biologically inert, and optically transparent surface that will
allow cells to attach and mouve - polystyrene plastic -
supplied sterile and disposable
3
 Primary Culture
• When cells are surgically removed from an organism and
placed into a suitable culture environment, they will attach,
divide and grow.
• There are two basic methods.
– Explant Cultures, small pieces of tissue are attached to a culture
vessel and bathed in culture medium. After a few days, individual
cells will move from the tissue explant out onto the culture vessel
surface
– Enzymatic Dissociation, uses digesting (proteolytic) enzymes,
such as trypsin or collagenase, to dissolve the cement holding the
cells together - a suspension of single cells that are then placed
into culture vessels containing culture medium

4
 Cell culture phases
• Lag Phase - usually the first 1-2 days, there is little or no increase
in cell number. The cells undergo internal cytoskeletal and enzyme
changes and adjust to the new media.
• Log Phase - the cell number increases exponentially. This growth
will continue as long as there are sufficient nutrients to support the
increasing cell number. Eventually some critical nutrient will become limiting,
however.
• Plateau Phase - the number of cells remains constant. Eventually,
the cells will die unless subcultured or fresh media is added. The cells
will continue to grow in contact with the vessel and give rise to a
"monolayer" culture. The cells will cease to divide when they reach
confluency; they demonstrate contact inhibition.

5
 Subculturing
• Ideally, cells are harvested when they are in a semi-
confluent state and are still in log phase
• This is usually done by removing them as gently as possible
from the substrate with enzymes.
• Some cell lines can be harvested by gently scraping the
cells off from the bottom of the culture vessel.
• Once released, the cell suspension can then be subdivided
and placed into new culture vessels.
• Once a surplus of cells is available, they can be treated with
suitable cryoprotective agents, such as dimethylsulfoxide
(DMSO), carefully frozen and then stored at cryogenic
temperatures (below -130°C) 6
 Media and growth requirements
• Physiological parameters
– pH - 7.2-7.5 and osmolality of medium must be maintained
– temperature - 37ºC
– humidity is required
– gas phase - bicarbonate conc. and CO2 tension in equilibrium
– Away from visible light - light induces production of toxic compounds in some media;
• Medium requirements: (often empirical)
– Ions - Na, K, Ca, Mg, Cl, P, Bicarbonate
– Trace elements - iron, zinc, selenium
– sugars - glucose is the most common
– amino acids - essential
– vitamins - B, etc.
– choline, inositol
– serum - contains a large number of growth promoting activities such as buffering toxic nutrients by
binding them, neutralizes trypsin and other proteases, has undefined effects on the interaction
between cells and substrate, and contains peptide hormones or hormone-like growth factors that
promote healthy growth.
– antibiotics - not required for cell growth, antibiotics are often used to control the growth of
bacterial and fungal contaminants.

• Feeding - 2-3 times/week. 7

8
 Cell Fractionation
• chemical knowledge of organelle function by isolating
organelles into reasonably pure fractions
• Each organelle has characteristics (size, shape and
density for example) which make it different from other
organelles within the same cell
• The process of breaking open cells is homogenization
• the subsequent isolation of organelles is fractionation

9
 Homogenization
• Cells which are part of a more solid tissue (such as liver or
kidney) will first need to be separated from all connections
with other cells
• need to be enzymatically or mechanically disaggregated
• Homogenization techniques can be divided into
– those brought about by osmotic alteration of the media which cells
are found in
– those which require physical force to disrupt cell structure : use of
blenders, or ultrasonification

10
 Osmotic alterations
• Many organelles are easier to separate if the cells are
slightly swollen.
• The inbibition of water into a cell will cause osmotic
swelling of the cell - rupture of the cell membrane and
subsequent organelle separation.
• The use of a hypo-osmotic buffer can be very
beneficial, for example, in the isolation of mitochondria
and in the isolation of mitotic chromosomes.

11
 Ultrasonication
• Ultrasonicators have been used to separate organelles
from cells, particularly from tissue culture cells.
• Light use of an ultrasonic wave can readily remove
cells from a tissue culture substrate (such as the
culture flask).
• It can also be adjusted to separate cells, or to break
open the plasma membrane and leave the internal
organelles intact

12
Cell fractionation allows the isolation of cell
constituents by differential centrifugation.
The drawings at right show the cellular organelles at the
bottom of each tube after centrifugation. Centrifugal force is
expressed by g, which is equivalent to the force of gravity.
(1) A fragment of tissue is minced and
dissociated with a homogenizer or by
ultrasound.
(2) The dissociated tissue is left standing for
about 20 min. Clumps of cells and fibers of
extracellular matrix precipitate to the bottom.
(3) The supernatant is centrifuged at 1000 g for
20 min. Nuclei precipitate.
(4) The supernatant is centrifuged at 10,000 g
for 20 min. Mitochondria and lysosomes
precipitate.
(5) The supernatant is centrifuged at 105,000 g
for 120 min. Microsomes precipitate.
(6) If the supernatant is first treated with sodium
deoxycholate and then centrifuged at 105,000
g for 120 min, the microsomes dissociate and
precipitate separately as endoplasmic
13
reticulum membranes and ribosomes.
Electron micrographs of 3 cell fractions isolated by density gradient centrifugation.
A: Mitochondrial fraction, contaminated with microsomes. B: Microsomal fraction.
C: Lysosomal fraction. High magnifications. (Courtesy of P Baudhuin.)
14
 PROTEIN
ISOLATION

15
Ultracentrifugation (A) and chromatography (B):
methods of protein isolation.
A: A mixture of proteins obtained from homogenized
cells or tissues is submitted to centrifugation at high
speed for several hours. The proteins separate into
several bands, depending on the size and density of
the protein molecules. The ultracentrifugation medium
is drained and collected in several fractions that
contain different proteins, which can be analyzed
further.
B: A mixture of proteins obtained from homogenized
cells or tissues is added to a column filled with
particles that have different chemical properties -
different electrostatic charges (attracting proteins
according to their charge) or different sizes of pores
(acting as sieves for different-sized molecules). As the
proteins migrate through the column, their movement
is slowed according to their interaction with the
particles. When the effluent is recovered, the different
groups of proteins may be collected separately.
16
Ion exchange chromatography
• based on the charge of the protein you are trying to isolate.

• If the protein has a high positive charge, you'll pass it through a

column with a negative charge or, you can bind a negatively charged
protein to a positively charge column
• The charge on the column will bind the charged protein, and other
proteins will pass through.
• to release your positively charged protein from the negatively
charged column - a cation exchange column uses sulfonated
residues.
• positively charge column is called an anion exchange column -
uses quaternary ammonium residues.

17
 Gel filtration chromatography
• separates proteins on the basis of their size. The column is
packed with a matrix of fine porous beads
• The beads have very small holes.

• As the protein solution is poured on the column, small

molecules enter the pores in the beads.

• Larger molecules are excluded from the holes, and pass

quickly between the beads

18
19
Gel electrophoresis: a method of protein isolation.
A: Isolation of proteins.
(1) Mixtures of proteins are obtained from homogenized
cells or tissues. They are usually treated with a strong
detergent (sodium dodecyl sulfate) and with
mercaptoethanol to unfold and separate the protein
subunits.
(2) The samples are put on top of a polyacrylamide gel,
which is submitted to an electrical field. The proteins
migrate along the gel according to their size and shape.
(3) A mixture of proteins of known molecular mass is added
to the gel as a reference to identify the molecular mass of
the other proteins.
B: Detection and identification of the proteins.
(1) Staining. All proteins will stain the same color. The
color intensity is proportional to the protein concentration.
(2) Autoradiography. Radioactive proteins can be
detected by autoradiography. An x-ray film is apposed to
the gel for a certain time and then developed. Radioactive
proteins will appear as dark bands in the film.
(3) Immunoblotting. The proteins can be transferred from
the gel to a nitrocellulose membrane. The membrane is
incubated with an antibody made against proteins that may
be present in the sample. 20
 SDS-PAGE
• SDS-PAGE stands for sodium dodecyl sulfate-polyacrylamide gel
electrophoresis

• SDS portion it is an anionic detergent that binds quantitatively to

proteins, giving them linearity and uniform charge, so that they can
be separated solely on the basis of their size

• The number of SDS molecules that bind to a protein is proportional to

the number of amino acids that make up the protein.

• Each SDS molecule contributes two negative charges, overwhelming

any charge the protein may have.

• SDS also disrupts the forces that contribute to protein folding,

ensuring that the protein is not only uniformly negatively charged, but
linear as well
21
• polyacrylamide gel electrophoresis separates protein molecules
according to their size.
• is a cross-linked matrix that functions as a sort of sieve to help "catch"
the molecules
• an electric current is used to move the protein molecules across a
polyacrylamide gel.
• The smaller molecules are able to navigate faster than the larger one,
so they make it further down the gel
• Once an SDS-PAGE gel is run, fix the proteins in the gel so they don't
come out when you stain the gel. Acetic acid 25% in water is a good
fixative, as it keeps the proteins denatured.
• The gel is typically stained with Coomasie blue dye R250

22
 Western Blot
• Western blot analysis can detect one protein in a
mixture of proteins while giving you information
about the size of the protein.

• Western blotting tells you how much protein has

accumulated in cells

23
• Separate the proteins by size using SDS-PAGE
• Place a nitrocellulose membrane on the gel and, using
electrophoresis, drive the protein bands onto the
nitrocellulose membrane drive the negatively charged proteins over to the
positively charged nitrocellulose membrane

• Incubate the nitrocellulose membrane with a primary

antibody
• Incubate with a secondary antibody. This antibody should be an
antibody-enzyme conjugate - alkaline phosphatase
• To see the enzyme in action, incubate it with a substrate
which will precipitate
• Put x-ray film on your gel to detect a flash of light, which
is given off by the enzyme

24
25
 RIP (radio-immune
precipitation)
• If you are more interested in the rate of synthesis of
protein in a cell, or if your protein degrades too
quickly to be detected by a Western blot
• also detects protein-protein interaction, while
Western blotting can't.
• Uses radioactivity

26
 ELISA (Enzyme-Linked
Immunosorbent Assay)
• powerful method in estimating ng/ml to pg/ml of
peptides, proteins, antibodies and hormones in the
solution, such as serum, urine and culture supernatant.
• an antigen must be immobilized to a solid surface.
• The antigen is then recognized by an antibody that is
linked to an enzyme.
• Detection is accomplished by incubating the enzyme-
complex with a substrate that produces a detectable
product.

27
• Most commonly, ELISAs are performed in 96-well (or 384-
well) polystyrene plates

• The antigen is added to the wells where some remain

adsorbed by hydrophobic association to the walls after
washing away the excess

• During an infection, an individual mounts an antibody

response - production of plasma IgG molecules that bind to
various parts of the infectious agent.

• If these antibodies are present in the sample, they will bind

to the adsorbed antigens in the well and remain there after
washing 28
29
30
NUCLEIC ACIDS
ANALYZE

31
 Southern blotting
• named after Edward M. Southern who developed this
procedure at Edinburgh University in the 1970s
• DNA molecules are transferred from an agarose gel onto a
membrane.
• locates a particular sequence of DNA within a complex
mixture. For example, Southern Blotting could be used to locate a particular gene within
an entire genome.
• The amount of DNA needed for this technique is dependent
on the size and specific activity of the probe. Short probes tend to
be more specific

32
33
• Digest the DNA with an appropriate restriction enzyme.
• Run the digest on an agarose gel
• Denature the DNA separate  double-stranded DNA into single-
stranded DNA
• Transfer the denatured DNA to the membrane. a
nitrocellulose membrane. Transfer is usually done by capillary action
• treat it with UV light. This cross links (via covalent bonds) the
DNA to the membrane.
• Probe the membrane with labeled ssDNA. Hybridization -
ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of
complementary strands.
• Visualize the radioactively labeled target sequence by
autoradiograph

34
 Northern blotting
• locating a sequence of RNA

• Northern hybridization or RNA hybridization

• The procedure is almost identical to that of

Southern blotting, except you are working with
RNA instead of DNA

35
 PCR (polymerase chain
reaction)
• a biological sample with trace amounts of DNA in it. You
want to work with the DNA, characterize it by sequencing
• PCR is the amplification of a small amount of DNA into a
larger amount.
• It is quick, easy, and automated
• developed by Nobel laureate biochemist Kary Mullis in
1984 and
• based on the discovery of the biological activity at high
temperatures of DNA polymerases found in thermophiles

36
• Most DNA polymerases (enzymes that make new DNA)
work only at low temperatures. But at low temperatures,
DNA is tightly coiled

• thermophile DNA polymerases, called Taq polymerase,

work at 100C, a temperature at which DNA is denatured (in
linear form - named after Thermus aquaticus

• PCR is so efficient because it multiplies the DNA

exponentially for each of the 25 to 75 cycles. A cycle takes only a
minute or so and each new segment of DNA that is made can serve as a template for
new ones

37
 4 things to perform PCR

• The target sample. the DNA sample.

• A primer. Short strands of DNA that adhere to the target
segment. They identify the portion of DNA to be
multiplied and provide a starting place for replication.
• Taq polymerase. This is the enzyme that is in charge of
replicating DNA. This is the polymerase part
• Nucleotides. (dNTPs) so the DNA polymerase has
building blocks to work with.

38
 3 major steps repeated 25 to 75
times
• Target sample is heated. This denatures the DNA, unwinding it and
breaking the bonds that hold together the two strands - single stranded
DNA (ssDNA).

• Temperature is reduced and the primer is added. The primer

molecules now have the opportunity to bind (anneal) to the pieces of
ssDNA. This labels the portions of DNA to be amplified and provides a starting place for
replication.

• New pieces of ssDNA are made. Taq polymerase catalyzes the

generation of new pieces of ssDNA that are complimentary to the
portions marked by the primers. This is the chain reaction in the name polymerase
chain reaction.

39
40
• Then run an Agarose gel electroporesis to visualize the
fragments
• separates different DNA molecules according to their size.
• The phosphate molecules that make up the backbone of
DNA molecules have a high negative charge.
• When DNA is placed on a field with an electric current,
these negatively charged DNA molecules migrate toward
the positive end of the field
• The smaller molecules are able to navigate the mesh faster
• The gel is stained with ethidium bromide so you can
visualize how these DNA molecules resolved into bands
along the gel

41
 RT-PCR
• The incorporation of the enzyme reverse transcriptase (RT), can be
combined with traditional PCR to allow for the amplification of RNA
molecules.

• add your RNA sample to the PCR machine,

• add a DNA primer as usual and allow it to anneal to your target

molecule.

• add RT along with dNTPs, which will elongate the DNA primer and
make a cDNA copy of the RNA molecules

• run the PRC reaction as usual.

• The product of RT-PCR is a double stranded DNA molecule analogous

to the target segment of the RNA molecule. 42