purpose of components in biological solutions. [email protected] this talk is about: how lab...
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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
MANY TYPES OF SOLUTIONS
Solutions differ for different molecules Proteins Nucleic acids Membrane structures Intact cells Etc.
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
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)
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
FOCUS ON
Structure and function of proteins and nucleic acids in solution
Talk about a few important components of solutions
PROTEINS
Many functions in cells Enzymes Antibodies Transcription factors Transporting agents Etc.
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
PRIMARY STRUCTURE
Linear sequence of amino acids Peptide bonds hold amino acids together Beads on a string Peptide bonds are covalent
Strong bonds
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
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
PRIMARY STRUCTURE
Linear polymer of nucleotide subunits Connected into strands by covalent
phosphodiester bonds. Strong bonds, primary structure.
SECONDARY STRUCTURE
Double-stranded Complementary pairs of bases are held
together by hydrogen bonds Relatively weak
RNA
RNA (ribonucleic acid) also is a polymer of nucleotides
Single-stranded and shorter than chromosomal DNA.
SECONDARY STRUCTURE
Sometimes complementary bases within an RNA strand pair
Weak interactions cause RNA to fold into various conformations
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
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
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
TERM “BUFFER”
Term “buffer” may refer just to buffering agent, or to entire solution
ALSO ADD
Salts Reducing agents that prevent unwanted
disulfide bonds in proteins -DTT or beta-ME
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
SO,
May or may not preserve the higher order structure of biological molecules in solutions.
What about primary structure?
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
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
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
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.
SO,
Strong protein denaturing agents are used to destroy RNases 6 M urea SDS Guanidinium salts
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
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
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
Nucleases may be added to nucleic acid solutions to perform a particular task. restriction endonucleases
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.
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
ANOTHER EXAMPLE:
Detergents can make some membrane-associated proteins go into solution Usually use nonionic detergents Solubilizing agent
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
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
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
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
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.)
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.
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.
SUMMARY
Buffers Salts Proteases/nucleases Cofactors Detergents Organic solvents Solubilizing agents Denaturing agents Precipitating agents Reducing agents Metal chelators Anti-microbial agents Protease inhibitors
Living systems are aqueous Often need very high quality water
Cell culture Analytical methods Pharmaceutical products
BIOTECH COMPANIES
Purified water is a major expense in company May be most expensive raw material
PURIFICATION METHODS
Distillation Water purification systems
Reverse osmosis Ion exchange Filtration Millipore systems well-known
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.
SOURCES OF WATER
House deionized, may be adequate for molecular biology
Distilled Purchase water Purchase a water purification system