Transcript MORE ABOUT SOLUTIONS - Bio-Link

PURPOSE OF COMPONENTS IN
BIOLOGICAL SOLUTIONS
THIS TALK IS ABOUT:



How lab solutions support biological activity
and/or structure
Why solutions have the components that they
do
Handling biological materials in solution
[email protected]
MANY TYPES OF SOLUTIONS

Solutions differ for different molecules





Proteins
Nucleic acids
Membrane structures
Intact cells
Etc.
[email protected]
SOLUTIONS DIFFER
DEPENDING ON PURPOSE
1. Maintain activity of molecule(s)
2. Separate and purify molecule(s)
3. Store molecule(s)
4. Test identity, nature, or quantity of
molecule(s)
5. Culture whole cells
[email protected]
EXAMPLES


Solutions for cutting DNA into fragments
(identity) may be different than for enzyme
activity (activity)
Extraction buffer (separation/purification)
different than storage buffer (storage)
[email protected]
WHAT IS THE PURPOSE OF
YOUR SOLUTION?
1. Maintain activity of molecule(s)
2. Separate and purify molecule(s)
3. Store molecule(s)
4. Test identity, nature, or quantity of
molecule(s)
5. Culture whole cells
[email protected]
FOCUS ON


Structure and function of proteins and nucleic
acids in solution
Talk about a few important components of
solutions
[email protected]
PROTEINS

Many functions in cells





Enzymes
Antibodies
Transcription factors
Transporting agents
Etc.
[email protected]
PROTEINS ARE DIVERSE IN
STRUCTURE



Proteins can do many things because they
are structurally diverse
Are polymers composed of 20 different amino
acid building blocks
Amino acids have different properties
[email protected]
PRIMARY STRUCTURE




Linear sequence of amino acids
Peptide bonds hold amino acids together
Beads on a string
Peptide bonds are covalent

Strong bonds
[email protected]
PROTEINS FOLD INTO
COMPLEX SHAPES





Proteins fold into specific 3-D shapes
Each protein’s shape depends in its amino
acid composition
Every protein consists of different amino
acids, so every protein has a different shape
Called “higher order structure”
Stabilized by weak interactions, such as
hydrogen bonds
[email protected]
STRUCTURE OF DNA

In many ways, DNA is structurally and
functionally simpler than protein


Only four different types of subunit, not 20
Always same shape, always double-stranded
helix
[email protected]
PRIMARY STRUCTURE


Linear polymer of nucleotide subunits
Connected into strands by covalent
phosphodiester bonds.

Strong bonds, primary structure.
[email protected]
SECONDARY STRUCTURE


Double-stranded
Complementary pairs of bases are held
together by hydrogen bonds

Relatively weak
[email protected]
RNA


RNA (ribonucleic acid) also is a polymer of
nucleotides
Single-stranded and shorter than
chromosomal DNA.
[email protected]
SECONDARY STRUCTURE


Sometimes complementary bases within an
RNA strand pair
Weak interactions cause RNA to fold into
various conformations
[email protected]
HIGHER ORDER STRUCTURE
IN NATURE


Higher order structure of proteins, DNA, and
RNA is held together by relatively “weak”
interactions
In nature, “weakness” is important


Enzymes change shape when bind their
substrates
DNA strands come apart in replication and
transcription
[email protected]
IMPLICATIONS IN LAB

Loss of higher order structure occurs fairly
easily





Affected by changes in pH
Ionic strength
Temperature
May or may not be reversible
Called denaturation
[email protected]
HIGHER ORDER STRUCTURE
IN LAB


Often manipulated in lab, depending on
purpose of solution
If purpose of solution is to sustain normal
function/activity, must protect structure
[email protected]
TO PROTECT STRUCTURE

Add:

Buffering agents
 Tris
 Phosphate
 HEPES
 PIPES
[email protected]
TERM “BUFFER”

Term “buffer” may refer just to buffering
agent, or to entire solution
[email protected]
ALSO ADD


Salts
Reducing agents that prevent unwanted
disulfide bonds in proteins -DTT or beta-ME
[email protected]
OTHER TIMES


But, solution may have other purposes
Denature higher order structure with
detergents and other denaturants


Destroy folding when we do PAGE with SDS
Phenol and chloroform denature proteins during
DNA isolation
[email protected]
SO,

May or may not preserve the higher order
structure of biological molecules in solutions.

What about primary structure?
[email protected]
PRIMARY STRUCTURE IN NATURE



Primary structure harder to disrupt
If disrupted, destroy the molecule
Can be broken apart by enzymes that digest
the covalent bonds



Proteases and nucleases
Occurs naturally in digestion
Occurs naturally in cells, recycling
[email protected]
IN LAB



Proteases and nucleases often a problem
Might come from bacteria, or disrupted cells, or skin
from people
Sometimes add anti-microbial agents to solutions



Sodium azide
Might add anti-protease agents
Usually store solutions in the cold
[email protected]
ALSO USE CHELATORS


DNA degrading nucleases require Mg++ as a
cofactor
EDTA is often added to nucleic acid solutions
to chelate magnesium and remove it from
solution.

TE buffer, protect DNA structure and function
 Tris buffer, control pH
 EDTA chelating agent
[email protected]
RNA NUCLEASES

RNA nucleases are special problem




Ubiquitous
Difficult to destroy
Generally do not require metal ion cofactors to be
active.
RNase A, can even survive periods of boiling or
autoclaving.
[email protected]
SO,

Strong protein denaturing agents are used to
destroy RNases



6 M urea
SDS
Guanidinium salts
[email protected]
ALSO


RNA nucleases frequently contaminate
glassware and other laboratory items
Hands are a major source of RNase
contamination; gloves should be worn when
working with RNA

Wear gloves to protect product and not people
[email protected]

Once gloves have come in contact with a
surface that was touched by skin (for
example, a pen, notebook, laboratory bench,
etc.) the gloves should be changed
[email protected]
BUT, PROTEASES AND
NUCLEASES


May be added to solutions intentionally
When working with DNA, common to add
proteases, like proteinase K

To destroy endogenous nucleases
[email protected]

Nucleases may be added to nucleic acid
solutions to perform a particular task.

restriction endonucleases
[email protected]
PRECIPITANTS

Ethanol plays an important role in working
with nucleic acids because it precipitates
DNA and RNA.

Nucleic acids do not lose their structural or
functional integrity when isolated with
phenol/chloroform and/or ethanol.
[email protected]
DETERGENTS

Ionic detergents have hydrophilic portions
that are ionized in solution


SDS (sodium dodecyl sulfate) is an example
Others have hydrophilic sections that are not
ionized in solution, nonionic detergents

Triton X-100 is a nonionic detergent
[email protected]
ANOTHER EXAMPLE:

Detergents can make some membraneassociated proteins go into solution


Usually use nonionic detergents
Solubilizing agent
[email protected]
OSMOTIC STABILIZER

Maintain osmotic equilibrium



Glucose
Gelatin
Salts
[email protected]
SALTS



Life evolved in the sea; salts perform
essential roles in organisms
Salt levels are rigorously controlled in nature
Must be controlled in lab solutions
[email protected]


Salts affect charges on proteins and DNA
Modify




Higher order structure
Solubility
Binding of biological molecules to one another
Binding of biological molecules to matrices
[email protected]
EXAMPLE: PROTEIN
SOLUBILITY

Salts affect protein solubility

Manipulate to keep proteins in solution
 Manipulate to cause them to precipitate
Used in purification schemes for proteins

[email protected]
SALTS AND NUCLEIC ACIDS


Hybridization is binding of single-stranded
DNA with short strands of complementary
DNA or RNA
Is affected by ionic strength of the solution
[email protected]
SALTS AND STINGENCY

Stringency relates to reaction conditions
when single-stranded nucleic acids are
allowed to hybridize

High stringency: binding occurs only between
strands with perfect complementarity. (Every
guanine is base-paired with a cytosine and
every adenine is base-paired with a thymine.)
[email protected]

At lower stringency, there can be some
mismatch of bases across the strands and
hybridization still occurs.

Situations where high stringency is required
and other situations where lower stringency is
desirable.
[email protected]
SALT AND STRINGENCY

Low stringency conditions: salt concentration
is high and the temperature is relatively low.
Can be some mismatches.

High stringency: when temperature is higher
and salt concentration is lower, must match
exactly.
[email protected]
SUMMARY













Buffers
Salts
Proteases/nucleases
Cofactors
Detergents
Organic solvents
Solubilizing agents
Denaturing agents
Precipitating agents
Reducing agents
Metal chelators
Anti-microbial agents
Protease inhibitors
[email protected]
MOST IMPORTANT COMPONENT IN
ANY SOLUTION IS

WATER
[email protected]


Living systems are aqueous
Often need very high quality water



Cell culture
Analytical methods
Pharmaceutical products
[email protected]
BIOTECH COMPANIES


Purified water is a major expense in company
May be most expensive raw material
[email protected]
PURIFICATION METHODS


Distillation
Water purification systems




Reverse osmosis
Ion exchange
Filtration
Millipore systems well-known
[email protected]
HOWEVER…

Regardless of method used, no such thing as
“pure” water
[email protected]
CONTAMINANTS




Excellent solvent, dissolves contaminants from a
wide variety of sources.
More pure, the more aggressive it is
Contaminants may leach into water from glass,
plastic, and metal containers.
If water is not sterilized, microorganisms readily
grow in it and may release toxic bacterial
byproducts.
[email protected]
SOURCES OF WATER




House deionized, may be adequate for
molecular biology
Distilled
Purchase water
Purchase a water purification system
[email protected]