Tools of Cell Biology Flashcards
What are antibodies, how are they made, how are they used?
-Techniques include:
- Immunopreciptiation
- Immunochemistry
- ELISA
- Flow cytometry
- Western blotting
- Homogenization of cells
- Centrifugation
Is a big Quaternary protein that has a variable region (arms) and a constant region. Both of these regions are recognizable and bind to appropriate factors.
Antibodies (Ab)
SEE NOTES FOR DIAGRAM
Antibody specificity; part of antigen recognized by antibody. (antigen can have many of these)
- usually, one arm of the antibody will bind to this.
- Structure and fxn are vital (if structure is changed, binding will not occur)
Epitope
Foreign protein (particle) that the antibody recognizes
Antigen
Region(s) of the antigen to which the antibody binds.
Epitope
SEE NOTES FOR DRAWN DIAGRAM
_____ cells produce anitbodies and each is unique (has its own antibodies).
-Are made by bone marrow
B
“Normal” role of antibodies
- Antibodies cross-link antigens into aggregates
- Antibody-antigen aggregates are ingested by phagocytic cells and special proteins in blood kill antibody coated bacteria or viruses.
* To recognize foreign proteins and trigger an immune response
How can you make antibodies that are specific to your protein of interest?
SEE NOTES FOR DRAWN DIAGRAM
- Purify your antigen of interest
- Choose an animal (SEE NOTES FOR OPTIONS TO USE)
- Inject animal with antigen
- Allow animal to make an immune response
- Perform a blood-draw on the animal (use plasma b/c antibodies like to hang out in the serum)
Single antibody recognizes a single epitope
Monoclonal Antibodies
Antibodies that can bind to different epitopes
Polyclonal antibodies
- B cell from animal injected with antigen A makes anti-A antibody but doesn’t divide forever.
- Tumor cells in culture divide indefinitely but do not make antibody.
- Fuse antibody-secreting B cell with tumor cell
- Hybrid cell makes and secretes antibody and divides indefinitely
How to make Monoclonal Antibodies
Monoclonal Antibodies: more information
- Benefit: can have for forever
- In order to isolate B cells, you need to kill the animal, screen to find the cells you want (in the spleen b/c that’s where B cells hang out), and it’s time intensive & expensive!
Review of Polyclonal Ab
- “inexpensive”
- limited time/ quantity of Ab
- multiple recognition of epitopes
- batch-to-batch variation
Review of Monoclonal Ab
- expensive (~$400 a mL)
- infinite supply
- recognition of only 1 epitope
- constant/ renewable
How can we use antibodies to study cells/proteins?
- where is a protein being expressed?
- find proteins/purify them
- regulate amount of protein product thus regulate gene expression
- study levels of proteins/use as a means of comparison
- from isolation, you can study structure and fxn
- linking antibodies to radioactivity (novel drug development)
Is used to isolate a protein of interest and maybe other proteins that bind to it by:
- conjugating an antibody to bead
- add cell lysate to column
- antibody binds protein
- wash column
- elute off protein of interest so that it’s specifically isolated
Immunopreciptiation
Immunopreciptiation: How do you get the antibody off the protein?
- change pH to affect ionic interactions
- heat to denature, which will denature everything
- use urea b/c it mimics the protein and boots off the Ab by overwhelming it.
Using antibody to answer questions as where proteins are bound, how much protein is there, and is there a presence of protein?
- looking inside the cell (cyto)
- looking in tissue (histo)
Immunocytochemistry (ICC) and Immunohistochemistry (IHC)
SEE NOTES FOR IMPORTANT DETAILS!!
Enzyme-linked Immunosorbent assay
- a lot of medical tests are done with this method
- test for a color change
ELISA
Uses antigen to detect Ab
ex. HIV testing
Indirect ELISA
Can use Ab to detect antigen
ex. contamination tests done on food
Direct ELISA
Steps for ELISA direct
- antibody is absorbed onto the well and sensitizes the plate.
- test antigen is added; if complementary, antigen binds to antibody
- wash—->enzyme-linked antibody specific for test antigen (enzyme w/ color label) then binds to antigen, forming a double antibody sandwich
- wash—>enzyme’s substrate is added, and rxn produces a visible color change that is measured spectrophotometrically
Steps for ELISA indirect
- Antigen is absorbed onto the well and sensitizes the plate.
- Test antiserum is added; if antibody is complementary, it binds to the antigen
- wash—>enzyme-linked anti-gamma globulin (secondary Ab) binds to bound antibody
- wash—> enzyme’s substrate is added, and rxn produces a visible color change that is measured spectrophotometrically
Method that counts cells that you’ve labelled.
Flow Cytometry
