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Bio 234 > Tools of Cell Biology > Flashcards

Tools of Cell Biology Flashcards

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Biology 101 flashcards
1
Q

What are antibodies, how are they made, how are they used?

-Techniques include:

A
  1. Immunopreciptiation
  2. Immunochemistry
  3. ELISA
  4. Flow cytometry
  5. Western blotting
  6. Homogenization of cells
  7. Centrifugation
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2
Q

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.

A

Antibodies (Ab)

SEE NOTES FOR DIAGRAM

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3
Q

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)
A

Epitope

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4
Q

Foreign protein (particle) that the antibody recognizes

A

Antigen

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5
Q

Region(s) of the antigen to which the antibody binds.

A

Epitope

SEE NOTES FOR DRAWN DIAGRAM

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6
Q

_____ cells produce anitbodies and each is unique (has its own antibodies).
-Are made by bone marrow

A

B

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7
Q

“Normal” role of antibodies

A
  1. Antibodies cross-link antigens into aggregates
  2. 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
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8
Q

How can you make antibodies that are specific to your protein of interest?
SEE NOTES FOR DRAWN DIAGRAM

A
  1. Purify your antigen of interest
  2. Choose an animal (SEE NOTES FOR OPTIONS TO USE)
  3. Inject animal with antigen
  4. Allow animal to make an immune response
  5. Perform a blood-draw on the animal (use plasma b/c antibodies like to hang out in the serum)
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9
Q

Single antibody recognizes a single epitope

A

Monoclonal Antibodies

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10
Q

Antibodies that can bind to different epitopes

A

Polyclonal antibodies

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11
Q
  • 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
A

How to make Monoclonal Antibodies

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12
Q

Monoclonal Antibodies: more information

A
  • 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!
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13
Q

Review of Polyclonal Ab

A
  • “inexpensive”
  • limited time/ quantity of Ab
  • multiple recognition of epitopes
  • batch-to-batch variation
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14
Q

Review of Monoclonal Ab

A
  • expensive (~$400 a mL)
  • infinite supply
  • recognition of only 1 epitope
  • constant/ renewable
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15
Q

How can we use antibodies to study cells/proteins?

A
  • 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)
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16
Q

Is used to isolate a protein of interest and maybe other proteins that bind to it by:

  1. conjugating an antibody to bead
  2. add cell lysate to column
  3. antibody binds protein
  4. wash column
  5. elute off protein of interest so that it’s specifically isolated
A

Immunopreciptiation

17
Q

Immunopreciptiation: How do you get the antibody off the protein?

A
  1. change pH to affect ionic interactions
  2. heat to denature, which will denature everything
  3. use urea b/c it mimics the protein and boots off the Ab by overwhelming it.
18
Q

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)
A

Immunocytochemistry (ICC) and Immunohistochemistry (IHC)

SEE NOTES FOR IMPORTANT DETAILS!!

19
Q

Enzyme-linked Immunosorbent assay

  • a lot of medical tests are done with this method
  • test for a color change
A

ELISA

20
Q

Uses antigen to detect Ab

ex. HIV testing

A

Indirect ELISA

21
Q

Can use Ab to detect antigen

ex. contamination tests done on food

A

Direct ELISA

22
Q

Steps for ELISA direct

A
  1. antibody is absorbed onto the well and sensitizes the plate.
  2. test antigen is added; if complementary, antigen binds to antibody
  3. wash—->enzyme-linked antibody specific for test antigen (enzyme w/ color label) then binds to antigen, forming a double antibody sandwich
  4. wash—>enzyme’s substrate is added, and rxn produces a visible color change that is measured spectrophotometrically
23
Q

Steps for ELISA indirect

A
  1. Antigen is absorbed onto the well and sensitizes the plate.
  2. Test antiserum is added; if antibody is complementary, it binds to the antigen
  3. wash—>enzyme-linked anti-gamma globulin (secondary Ab) binds to bound antibody
  4. wash—> enzyme’s substrate is added, and rxn produces a visible color change that is measured spectrophotometrically
24
Q

Method that counts cells that you’ve labelled.

A

Flow Cytometry

25
Q

Method that can not only count labelled cells, but can sort them into different tubes.

A

Fluorescence-activated cell sorting (FACS)

26
Q

Cell Breakage: many techniques require you to break open the cells (lyse). Include:

A

Sonication
Detergent
Homogenizer
Pressure cell

27
Q

Use of high frequency sound waves to break through cell membrane

A

Sonication

28
Q

Chemical method (SDS is commonly used) to break through the cell membrane

A

Detergent

29
Q

Mechanincal/physical method to break through the cell membrane

A

Homogenizer

30
Q

“French press” Forces cells through a small opening, which lyses cells.

A

Pressure Cell

31
Q

Way to separate homogenate into fractions

  • based on density of molecules in solution
  • spin speed can be changed to pull out different densities
A

Centrifugation

32
Q

Sedimentation Coefficient

A

Look at notes for diagram. Be able to read graph

33
Q

Centrifugation method that pulls out molecules of different densities separately.

A

Differential centrifugation

34
Q

Utilizes a stabilizing sucrose gradient

  • sample sediments to a layer that matches sucrose
  • this layers homogenate into slowly sedimenting and fast sedimenting layers
A

Velocity sedimentation

35
Q

Utilizes a steep sucrose gradient

  • sample is distributed throughout the sucrose density gradient
  • at equilibrium, components have migrated to a region in the gradient that matches their own density (from low buoyant to high buoyant density layers)
A

Equilibrium sedimentation (density gradient centrifugation)

Bio 234 (8 decks)

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