Term
| Western Blot (Immunoblot) |
|
Definition
Detect and quantify proteins that react with a specific antibody
Western blotting can be used to determine if a given antigen is present
Concentration of the antigenic proteins
Physiological state of a protein (phosphorylation)
Useful for medical testing (HIV, other viruses, Mad Cow’s Disease, Lyme Disease), although many recently replaced by other techniques such as PCR |
|
|
Term
|
Definition
Antibody – protein that binds to an epitope
Specificity – ability to bind to specific target with low background
Affinity – how tightly the Ab binds to the target
Polyclonal
Multiple epitopes, lower specificity, higher affinity, more tolerant of different fixation conditions
Produced by injection into animals
Monoclonal
Single epitope, high specificity, lower affinity, sensitive to fixation conditions for tissue specimens
Produced from hybridomas
In some circumstances the polyclonal Ab may be more useful than monoclonal Ab for immunohistochemical studies of tissues for morphological examination
Neither can be used inside living cells (150 kDa, 4 chains)
Nanobodies – camel, shark, llama antibodies – only 2 heavy chains, continuous binding site, 12 kDa, can be expressed inside of cells. Shark antibodies can be boiled or put in urea and remain active |
|
|
Term
|
Definition
SDS-PAGE of the sample proteins
The gel is removed from the gel sandwich and placed flat onto a nitrocellulose or PVDF membrane
A set of absorbant pads is used to support the gel and membrane as a sandwich, and the assembly is held in a supporting clamp
An electric field perpendicular to the sandwich forces the proteins to migrate out of the gel and onto the membrane to which they adhere
Because the membrane has free sites remaining, it is coated with a mixture of nonspecific proteins to block these free sites (dry milk)
The secondary antibody is usually coupled covalently to an enzyme that catalyzes a chromogenic reaction
Horseradish peroxidase (HRP)
Alkaline phosphatase
The presence of the secondary antibody can be assayed by immersing the membrane into the substrate of the coupled enzyme
Why two steps? Primary antibodies conjugated to enzymes are available |
|
|
Term
|
Definition
Nitrocellulose
Nitric acid + cellulose, used in gun
powder, photography, pregnancy tests
Binds amino acids
Established protocols
Cheaper
PVDF (Polyvinylidene Difluoride)
Reusable
Tougher
Higher binding capacity
Must be activated by methanol |
|
|
Term
|
Definition
The membrane is then soaked in a solution containing an antibody to the protein of interest (primary antibody)
Monoclonal or polyclonal
Conformational epitope – can be non-adjacent amino acids
Linear epitope – 6-10 adjacent amino acids
Best antibody – to a linear conformational epitope, but may depend on procedure
Western – sample was boiled, denatured
Immunoprecipitation or immunohistochemistry – sample in native conformation
Since all the protein-binding sites on the membrane are blocked, the antibody can adhere to the membrane only if it interacts with its specific antigen (barring nonspecific protein aggregation or binding) |
|
|
Term
| Choice of Primary Antibodies |
|
Definition
Purchase: whatever’s available, and cheap (GST)
Inject protein or epitope into a mouse, rabbit, goat, or other animal, purify it
Recombinant antibodies
Use an antibody specific to an epitope in your construct (GST, MBP, his-tag, GFP) |
|
|
Term
|
Definition
After any unbound primary antibody is rinsed off, the presence of the antibody is detected by introducing a secondary antibody that will react with any antibody from the same biological source as the primary antibody
Accomplished by injecting the constant (Fc) region into another species
If the primary antibody was from a goat, the secondary antibody could be a rabbit antibody raised against goat |
|
|
Term
|
Definition
Detection
Visible compound production (TMB)
Autoradiography
Chemiluminescence
Direct Infrared fluorescence
detection (0.6 pg to 2.5 ng) |
|
|
Term
|
Definition
Enzyme-Linked ImmunoSorbent Assay
Detect antigen or antibody in a sample
Replaced immunoassay (radioactive Ab)
Pregnancy test – antibodies for hCG |
|
|
Term
|
Definition
Immunoprecipitation – precipitating an antigen out of solution using an antibody as a purification step
Co-immunoprecipitation (pull-down) – binding an antibody to a protein on solution, hoping to pull-down other proteins in complex with it (protein-protein interactions)
Add agarose bead with bound antibody, add to a cell extract
SDS, Mass spec, Western for identification |
|
|
Term
|
Definition
|
|
Term
|
Definition
|
|
