Lectures 17, 18 Flashcards
What is measured for single gels?
What is gels relative mobility (Rf)?
Measurement of distance vs size
Gels relative mobility (Rf)= how fat your protein band goes compared to something fast (dye)
Plot Rf vs molecular mass (kD) gives a protein size standard curve
Slides 3-4 L17
What are urea denaturing gels?
Hearing and running samples in a gel made with 6 M urea allows for proteins to denature but retain their native charge
Still denatures like SDS, just doesn’t cost proteins and give them negative or positive charge, they keep native charge
Proteins run in this system will have their mobility affected by size and charge
Run at a specific pH to control protein charges
Example slide 7 L16
Compare SDS and urea gels on slide 8 L17
Okay
Urea takes charge into account while SDS only accounts for size so they will have different orders
What are native (non-denaturing) gels?
Mobility of proteins depends on size, charge, and conformation (shape)
Charge depends on the pH of the buffer used
Gels run substantially slower that SDS PAGE and some samples may not enter the gel based on charge at a specific pH
Slide 10 L17
What is 2 dimensional protein electrophoresis?
What are in dimensions 1 and 2?
Proteins separated based on 2 factors in 2 dimensions so we can better track “why did this sample run differently”
Slide 16-18 L17
Dimension 1: isoelectric focusing gels- requires gel with a non diffusing pH gradient (acid on one side, base on the other)
Proteins will separate by their overall pl
Slide 12-13 L17
Dimension 2: SDS PAGE- isoelectric focusing strips are treated to denature proteins and coat with SDS
Uniformly negative proteins are next resolved by size
Slide 14 L17
How can you identify if a certain band is present?
Using antibody binding reactions (low tech) or spot protein sequencing (high tech)
What are the 3 lines of defence in immunology?
- Intact skin, mucous membranes, normal microbiota
- Natural killer cells and phagocytic white blood cells
Inflammation
Fever
Antimicrobial substances - Specialized lymphocytes (T cells and B cells)
Antibodies
What are antibodies and their types?
Antibodies recognize and bind antigens and specifically they each bind a single epitope
IgM (first)
IgE (allergies) E for evil (IgE recognizes things that give allergies)
IgG (main)
IgA (secreted) two antibodies back to back
IgD (dispensable)
Slide 21-22 L17
How are antibodies diverse?
Immature B cells have several V, D, and J sequences
Random deletions in the light and heavy chains make unique antibodies
Billions of novel antibodies are possible in humans
Slide 23 L17
What are the two tests for B-cell clonal selection?
- Does the B cell make hose binding antibodies?
If yes, kill cell (clonal deletion) - Does the B-cell make useful antigen binding antibodies?
If yes, stimulate cell to divide
What is antibody titer?
Reinfection boosts the amount of antibodies produced
Memory B cells will retain specific Ab potential for a lifetime (or less)
Slide 6 L18
How are B-cells activated?
Naive B cells will be activated upon antigen exposure to form plasma cells or memory B cells They will also undergo class switching where DNA rearrangement allows IgM-making cells to convert to IgG or others
Slide 5 L18
What are the 5 protective mechanisms of binding antibodies to antigens?
Agglutination- reduces number of infectious units to be dealt with
Opsonization- coating antigen with antibody enhances phagocytosis
Neutralization- blocks adhesion of bacteria and viruses to mucosa and blocks attachment of toxin
Antibody dependant cell mediated cytotoxicity- antibodies attached to target cell cause destruction by eosinophils
Activation of complement- causes inflammation and cell lysis
What are antibodies in situ?
Created against a single antigen by injecting sample into a lab animal
- usually a protein, or polysaccharides, nucleus acids and haptens
Serum is collected and antibodies are purified for lab use
Secondary antibodies can be produced using the tail (Fc) from primary antibodies
Slide 10 L18
What are the 4 most common ways of obtaining antibodies?
- Isolate your protein (carb, lipid, nucleus acid, hapten) of interest and inject into a lab animal over weeks/months
- Send your protein of interest to a company
A few months and a few thousand dollars can get you plasma, purified antibodies, antibodies + animals or even clones antibody cells - Contacting a researcher who has already made the antibody
- Buy the antibody from a commercial supplier (of antigen is popular enough)
