Bio Lab Quiz #3 Flashcards
ELISA
ELISA: enzyme-linked immunosorbent assay
- It is a commonly used laboratory test to detect antibodies in the blood. An antibody is a protein produced by the body’s immune system when it detects harmful substances, called antigens.
immunosorbent = proteins adsorb to plastic wells in 2 ways
1. ab capture (ag bound to wells)
2. ag capture (ab bound to wells)
used to detect disease (HIV)
Immune System - Role:
Recognize intruders
Respond and protect body
Respond next time intruders encountered
Immune System - Types of Immunity:
1.Innate – born with some defense
2.Passive – from external source (mother to infant)
3.Acquired – specific response to foreign substance
– Humoral – antibodies in blood
– Cell mediated – cells that destroy infected cells
Immune System - Cells:
B cells: made in bone marrow → make antibodies
Macrophages: remove foreign molecules (antigens) from blood and present antigen at surface
T cells: made in thymus, recognize antigen on macrophages and kills cell
Immune System - Antibodies - Structure:
Structure: composed of 6 protein subunits: two myosin heavy chains & 4 myosin light chains
made by B cells with each B cell making a unique antibody that recognized a single epitope
Immune System - Antibodies - Epitope:
Epitope: is a short sequence of amino acids recognized by the antibody
Immune System - Antibodies - Five types of Antibodies:
Five types of Antibodies: IgG (most abundant), IgM (first responders), IgA (tears), IgE (allergic rx), and IgD
Immune System - Antibodies - Response to disease exposure:
Response to disease exposure:
immune system makes antibodies against disease antigen
first makes IgM (detected within one week)
IgGs after 3 weeks
second exposure is faster
Immune System - Antibodies - Immune system problems:
Immune system problems:
hypersensitive response – overreact to antigens (allergies)
immunodeficiency – can not mount an effective immune response
autoimmune diseases
Immune System - Antibodies - Making Antibodies:
Making antibodies:
Polyclonal
inject antigen into animal
antibodies made against several epitopes
Monoclonal
isolate individual B cells from mouse
B cell clones (only one epitope recognized)
but B cells do not last long in culture
fuse with immortalized tumor like cells
(hybrid or hybridoma)
Using Antibody Tools - Primary and Secondary
Molecule of interest is injected into primary animal model
Animal makes antibodies against the molecule
Antibodies are purified (primary antibody)
Antibodies from the first animal model are injected into a second animal model
The second animal produces antibodies against the first antibody (secondary antibody)
Western Blot is used to confirm ELISA results bc More sensitive and More specific
The secondary antibody is purified and conjugated to a colorimetric substrate or to an enzyme that can cleave a colorimetric compound
HIV
- HIV is an RNA Retrovirus
- Transmitted by exchange of body fluids, sharing needles, or blood transfusion
- Infects T-Cells in the immune system and thus destroys the immune system
- Flu-like symptoms within 1-2 months followed by latent period of up to 10 years
- HIV may have spread from an animal host to humans
- Treated but not cured by drugs which inhibit the action of HIV enzymes
- HIV can be detected by ELISA or western blot technology. (Both of which are developed using the basis of the mammalian immune system) ELISA tests are very quick. Western Blot tests are slower and more expensive and are used for confirmatory tests.
Detecting Antibodies in Serum - HIV
After 4-8 weeks of exposure to the HIV virus, the body will have produced a detectable level antibodies (immune response) against HIV
ELISA (HIV-Test) detects the presence of serum antibodies against HIV protein antigens
This is how HIV is detected in clinical laboratories
Most common AIDS test
What are the reagents?
A reagent is a substance used to create a chemical reaction
Purified Antigen: Chicken gamma globulin
Primary antibody (Serum Samples): Polyclonal anti-chicken antibody made by rabbits
Secondary antibody (enzyme-linked): Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish peroxidase (HRP)
Enzyme substrate: 3,3’,5,5’ – tetramethylbenzidine (TMB) – a colorless solution that when oxidized by HRP turns blue
Microplate Strips
Microplate strips are made of polystyrene
Hydrophobic side chains in amino acids bind to the polystyrene wells
No coating is needed
Wash Buffer
Wash buffer contains phosphate buffered saline (PBS) to keep antibodies in a stable environment that helps keep their structure
Also contains Tween 20: a nonionic detergent removes non-specifically bound proteins and coats wells that acts as a blocking agent to reduce background
Antibody will only bind to the simulated HIV antigen
MUSCLE PROTEINS Overview
Analyze protein profiles from a variety of fish
Polyacrylamide Gel Electrophoresis:
Break protein complexes into individual proteins
Separates protein samples based on size
Western Blot Analysis:
Transfer the proteins to a nitrocellulose membrane
Identify proteins by immunodetection using specific antibodies against the protein of interest
Protein Profiler/ Western Blotting - Proteins - Biochemical Similarities:
Biochemical Similarities:
proteins made up of amino acids
Traits are the result of protein structure and function
DNA codes for proteins that confer traits
DNA → RNA → Protein → Trait
The central dogma of biology is that DNA is transcribed into RNA and RNA is translated into protein (know this). However, there are many exceptions to this, including RNA that is not translated into protein and serves a function itself and RNA that is reverse transcribed into DNA such as in some viruses.
Protein Profiler/ Western Blotting - Proteins - Biochemical Differences:
Biochemical Differences:
Changes in DNA lead to proteins with:
Different functions
Novel traits
Positive, negative, or no effects
Genetic diversity provides pool for natural selection = evolution
Making Proteins
DNA → mRNA → tRNA → amino acid
Protein structure-proteins are made of amino acids. There are 20 different amino acids. Proteins are specific sequences of amino acids joined together.
Levels of Protein Organization:
Primary- the amino acid sequence
Secondary-alpha helix, beta sheet
Tertiary-3D folding (disulfide bridges between cysteines)
Quaternary- multiple subunits
Protein Size:
Size measured in kilodaltons (kD)
Dalton = mass of hydrogen molecule = 1.66 x 10-24 gram
Average amino acid = 110 daltons
Can undergo post-translational modification
- proteolytic cleavage
- Glycosylation
- phosphorylation
Can cause an apparent change in molecular weight without change in sequence
Proteins can undergo post-translational changes such as proteolytic cleavage, glycosylation and phosphorylation. Each of these modifications can cause an apparent change in molecular weight without a change in the protein sequence.
Muscle proteins - Muscle Structures:
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Myofibrils - basic contractile element of muscle
Bundled into muscle fibers with contractile subunit called sacromere.
Muscle proteins are required for locomotion and survival and are thus essential. Because they are so important they are conserved in evolution. Myofibrils are the basic contractile elements of muscle. They are bundled into muscle fibers. The contractile unit is the sarcomere.
Muscle Contains Proteins of Many Sizes
Muscle proteins - Sarcomere + Actin + Myosin:
Sarcomere are made of actin and myosin protein filaments. The sarcomeres shorten when myosin hydrolyzes ATP and slides along the actin.
Actin
- thin filaments
Myosin
- Thick filaments
- Breaks down ATP for muscle contraction
Actin
5% of total protein
–20% of vertebrate muscle mass
375 amino acids= 42 kD
Myosin
6 subunits
two heavy subunits (220 kD)
four light subunits (15-25 kD)
Muscle Contraction:
Hydrolysis of ATP causes myosin and actin filaments to slide past one another
Sarcomeres shorten
Muscle contracts
Sarcomere are made of actin and myosin protein filaments. The sarcomeres shorten when myosin hydrolyzes ATP and slides along the actin.
Western Blot/immunoblot procedure
The Western blot or immunoblot is a tool to identify specific proteins in a sample by molecular weight and antibody binding specificity. The Southern blot was the first technique developed by researcher Edwin Southern. The Northern blot was developed to analyze RNA and western blot to analyze protein. We will detect the myosin light chain using an antibody against that protein.
In sample buffer:
Tris buffer to provide appropriate pH
SDS (sodium dodecyl sulfate) detergent to dissolve proteins and give them a negative charge
Glycerol to make samples sink into wells
Bromophenol Blue dye to visualize samples
Dithiothreitol (DTT) dye to disrupt secondary structure
Heat Samples:
Heat Samples: Heating the samples denatures protein complex allowing the separation of individual proteins of size
How does SDS-PAGE Gel work?
Negatively charged proteins move to positive electrode
Smaller proteins move faster
Proteins separate by size
Why Use Polyacrylamide Gels to Separate Proteins?
Polyacrylamide gel has a tight matrix
Ideal for protein separation
Smaller pore size than agarose
Proteins much smaller than DNA
The Gel
Two phases
- Upper or stacking gel lines up proteins
- Lower or separating gel
Polyacrylamide Gel Analysis:
If we stained the gels as we did in the previous module, the gel would look like this (the bands would be blue). We will also use prestained standards with proteins of known size (called Kaleidoscope standards see later slide). The positive control, called actin and myosin on this gel is actually a rabbit skeletal muscle sample containing actin, myosin heavy chain and 3 different size myosin light chains. Our antibody will detect myosin light chain 1. This gel has 5 different fish samples. We will use catfish, cod, flounder, haddock, salmon, squid, swordfish and trout. We will use the myosin light chain for immunodetection and compare the size in the different species.
Steps - Transfer
Transfer Proteins from the gel to the nitrocellulose membrane
- 1 hour @ 100V
- Blotting buffer 1x Tris glycine with 20% methanol
Steps - Blocking Buffer
five
pbs
tween 20
Blocking Buffer
- Remove membrane from the blotting sandwich and immerse in 25 ml of blocking solution
- 5% non-fat milk: Prevents the primary antibody from binding randomly to the membrane
- Phosphate buffered saline (PBS): Provides the correct environment (pH, Salt) to maintain protein shape
- 0.025% Tween 20: non-ionic detergent that prevents non-specific binding of antibodies to the membrane
Steps - Add the Primary Antibody, Enzyme
Add the Primary Antibody. anti- myosin light-chain, Wash
- Discard blocking solution
- Pour 10ml of primary antibody onto the membrane and gently rock for 30 minutes
Primary antibody will bind to the myosin light-chain
- Quickly rinse membrane in 50 ml of wash buffer and discard the wash buffer
- Add 50 ml of wash leave for 10 minutes on the rocking platform
Add Enzyme-linked Secondary Antibody, Wash
- Discard wash solution
- Pour 10ml of the secondary antibody onto the membrane and gently rock for 30 minutes
- Secondary antibody will bind to the primary antibody
- Quickly rinse membrane in 50 ml of wash buffer and discard the wash buffer
- Add 50 ml of wash leave for 10 minutes on the rocking platform
Add Enzyme Substrate, Watch for Color
- Development
- Discard wash solution
- Add 10 ml of the enzyme substrate (HRP color detection reagent) onto the membrane
- Incubate for 10 minutes
- The colorimetric substrate is cleaved by the enzyme conjugated (attached) to the secondary antibody
Rinse and Store
- Rinse the developed membrane twice with distilled water and blot dry
- Air dry for 30min-1hr and store in lab notebook
Steps - Results:
Blot from unstained Gel
Blot from stained Gel
KaleidoscopePrestained Molecular Weight Standards
Evolutionary Relationships between proteins
Determine Size of Fish Proteins:
Different fish have different muscle protein profiles (diff size proteins, different number of proteins). More closely related species would share more proteins than distantly related species. This is because changes in muscle function may occur that are adaptive to different environments. For our gels we will not stain for all the proteins so could not do the activity shown here but I just wanted you to see this.
Molecular Mass Analysis With Semi-log Graph Paper
Each Fish Has a Distinct Set of Proteins
Phylogenetic Tree
What is the central dogma of Biology?
transcription, translation, and replication
DNA → RNA → Protein → Trait
What are 3 examples of post-translational changes that occur with proteins?
proteolytic cleavage
Glycosylation
phosphorylation
Why is 5% nonfat milk present in the blocking buffer used for Western blots?
Prevents the primary antibody from binding randomly to the membrane
Phosphate buffered saline (PBS): Provides the correct environment (pH, Salt) to maintain protein shape
Explain how the immune system responds when someone is exposed to a disease.
immune system makes antibodies against disease antigen
first makes IgM (detected within one week)
IgGs after 3 weeks
second exposure is faster
Role: Recognize intruders, Respond and protect body and Respond next time intruders encountered the body will have produced a detectable level antibodies (immune response)
How is a monoclonal antibody made?
Making antibodies -
solate individual B cells from mouse
B cell clones (only one epitope recognized)
but B cells do not last long in culture
fuse with immortalized tumor like cells
(hybrid or hybridoma)
south north west
Southern - DNA
Northern - RNA
Western - Protien detects myosin light chain (immunoblot) - tool to id spec protein in a sample by molcular weight and antibody binding
