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Cell Biology > Lecture 3 and 4: Cell Biology Techniques, Part I and II > Flashcards

Lecture 3 and 4: Cell Biology Techniques, Part I and II Flashcards

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1
Q

What is whole cell lysate?

A

collection of cells produces cell lysate which is single cell suspension of desired cells

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2
Q

How does whole cell lysate become cell lysate?

A

Through lysis buffer (or any detergent)

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3
Q

What is cell lysate/homogenate/extract?

A

Sample after adding lysis buffer for plasma membranes disrupted and sample has all cellular components

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4
Q

What is cell fractionation?

A

separate cellular components from one another and isolate organelles via centrifuge

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5
Q

Cell fractionation is not used to ______ but to _______

A

isolate specific proteins; segregate different cellular components from a cell lysate

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6
Q

Second higher speed centrifugation:

A

Pellet contains mitochondria, cytosol, and other organelles

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7
Q

Cell fractionation use centrifuge to spin at _____ to pul down heavier molecules

A

different speeds

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8
Q

First low speed centrifugation results:

A

Pellet contains nuclei
Supernatant contains cytosolic proteins and organelles

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9
Q

The purpose of cell fractionation is to

A

evaluate 3 types of proteins and reduce complexity of sample

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10
Q

3 types of proteins resulted in cell fractionation:

A
  1. Cell lysate: all proteins
  2. Nuclear proteins: proteins found only in nucleus
  3. Cytosolic proteins: all proteins outside of nucleus
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11
Q

What is SDS-Page?

A

Separates protein in a mixture by size and allows to analyze proteins in cell lysate or cell fractionation samples

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12
Q

SDS Page contains:

A

SDS: detergent that denatures proteins and coats proteins with negative charge
Beta-mercaptoethanol (BME): reducing agent that eliminates disulfide bridges

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13
Q

Why is negative charge by SDS important in gel electrophoresis?

A

negative charge pulls down protein and allow for all proteins to have uniform charge

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14
Q

After SDS-PAGE, what are the options to analyze?

A
  1. Coomassie blue
  2. Western blot
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15
Q

What is coomassie blue?

A

Stain to detect and visualize all proteins in gel as stain binds to basic amino acids

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16
Q

What is the purpose of coomassie blue?

A
  • ensure cell lysates from different samples have equal protein loading
  • determine if protein was successfully purified
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17
Q

How is Western blot technique performed?

A
  1. Use SDS Page gel to separate protein mixture in sample
  2. Transfer separated proteins from gel to membrane
  3. Primary antibody recognizes specific protein on membrane
  4. Secondary antibody binds to primary antibody and used for visualization, more signal on membrane indicates more protein in cell
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18
Q

Purpose of Western blot

A

Measuring amount of specific protein with antibodies

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19
Q

How are Coomassie blue and Western blot similar and different

A

Similiar: both analyze proteins after SDS PAGE
Differ: comasie is nonspecific stain while western blot is specific

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20
Q

What are antibodies useful for?

A

Useful as they recognize bacteria, viruses, and other foreign molecules that cause harm

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21
Q

What is protein structure of antibodies?

A
  • composed of 4 polypeptide chains with quaternary structure, consisting of 2 identical light and 2 identical heavy chains
  • have disulfide bridges helping with tertiary and quaternary structure (inter and intra disulfide chains)
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22
Q

Does an antibody have a binding site?

A

Has 2 binding sites (Vh and Vc) which can bind to an antigen

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23
Q

What is an antigen?

A

substance that stimulates immune response

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24
Q

How are antibodies naturally made?

A
  • B cells which are circulating around the body are immune cells that make antibodies
  • Each B cell makes unique antibody that recognzies specific antigen via antigen binding sites
  • Antigens bind to B cells, causing B cell to produce antibodies
  • When antibody binds to antigen, B cells are activated and divide and antibodies are secreted
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25
Q

What happens to secreted antibodies?

A

Bind to antigens and target them for destruction by immune cells

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26
Q

What are polyclonal antibodies

A

mixture of antibodies that bind to different places on same antigen

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27
Q

Process of making polyclonal antibody

A

Step 1.
- Antigen designed to stimulate production of antibody that can bind to it
- Antigen will find b cell for correct antigen binding site
- B cell will undergo differentiation and start secreting massive amounts of antibody into blood stream
Step 2:
- Collect blood and antibodies that do bind to antigen are purified

28
Q

What is the purpose of an immunoprecipitation (IP) ?

A

uses antibodies to purify protein complexes and assess protein-protein interactions

29
Q

Why are monoclonal antibodies ideal for drug treatment and scientific research?

A
  • Purified from hybrid cells which provide infinite supply of antibodies
  • Have low variability between batches to be ideal for research and drug treatment
30
Q

Process of making monoclonal antibodies

A

Step 1:
Inject animal with antigen to stimulate immune response of production of antibodies

Step 2:
Isolate B cells from spleen to get activated B cells which have been activated from antigen injections

Step 3:
Fuse B cells with myeloma cells to make hybrid cells
Identify which hybrid cells produce effective antibody

31
Q

How is an immunoprecipitation performed?

A
  • Complete IP and follow with SDS PAGE and western blot
    Step 1.
    Generate cell lysate
    Cross link proteins with formaldehyde to stabilize protein protein interactions
    Step 2:
    Add antibody conjugated to the bead and antibody binds to protein of interest
    Step 3:
    Use centrifuge to pull down bead so proteins not bound by antibody are discarded and proteins bound by antibody are purified
    Step 4:
    Reverse cross linking
    Use SDS PAGE and western blot to determine which proteins are interacting with protein of interest
32
Q

What is protein purification?

A

purifying thousands of proteins from cell lysate into one protein

33
Q

What is the trick of process of protein purification?

A

Giving protein of interest a handle bar with fusion protein

34
Q

Fusion protein

A

Genetically engineered protein that is used to isolate a specific protein
Can contain a tag

35
Q

GST Tag

A

GST protein attached to gene sequence to make fusion protein which is inserted into cells

36
Q

What is taken advantage of GST for protein purification?

A

GST bind to GSH with very high affinity

37
Q

Steps on GST tag used to purify specific protein from a cell lysate

A
  1. Create cell lysate containing mixture of cells including cells expressing fusion proteins
  2. Cell lysate is added to column that has beads covered with GSH
    Fusion proteins will stay at top of column and binds to beads with GSH
    All other nonfusion proteins will flow through and be at bottom of column
  3. After cell lysate is added to column with gluthathione or solution of gluthatione is added, proteins will bind to gluthathione to pull fusion proteins down column
38
Q

How is GST able to bind with glutathione after binding with GSH?

A

GST and GSH bind is not permanent due to being weak non covalent interaction

39
Q

How can coomassie blue be used for protein purificaiton?

A

Used to determine which sample has purified protein after purification

40
Q

How should 5 samples look like with coomassie blue after protein purification?

A

Each sample gets more puriifed
First sample/lane will have lots of bands
Second and third sample/lane will have less bands and a bit more fusion proteins
Fourth and fifth sample/ lane will have one distinguished band indicating purified protein

41
Q

Light microscope

A

Uses light source to view single cells and internal structure of a cell

42
Q

3 requirements for light microscope

A
  1. Light source must be focused on specimen
  2. Specimen must be transparent
  3. Set of lenses (objective and eyepiece) must focus the image
43
Q

Advantages of using light microscope

A

Easy and cheap to use
Able to do live imaging and view internal structures

44
Q

Disadvantages of using light microscope

A

Specimen must be transparent and colorless
Internal structures are difficult to see
Limit to magnification since light source is used

45
Q

Fluorescence Microscope

A

Use light source, but structures can be seen in greater detail with fluorescent labels

46
Q

Each fluorescence label has….

A

excitation and emission wavelength

47
Q

How does a fluorescent label work?

A
  1. First filter selects excitation wavelength that will excite label in specimen
  2. When wavelength reaches label, it will emit light at different wavelength and label comes back to ground state which releases photon
  3. Second filter selects wavelength emitted by label so photon can be detected, cause visualization while rest of specimen is black
48
Q

Advantage of fluorescence microscopy

A

Live imaging can be performed
3D structures by confocal

49
Q

Disadvantage of fluorescence microscopy

A

More expensive than light microscopy
Fluorescence signal will fade over time (photobleaching

50
Q

Short wavelength =

A

high energy

51
Q

Long wavelength =

A

low energy

52
Q

How do scientists attach fluorescent tags to specific proteins?

A

Fluorescence tags can be added to DNA sequence to create fluorescent protein (aka fusion protein)

53
Q

What is a common fluorescent tag attached to proteins?

A

GFP (green fluorescent protein)

54
Q

What is the purpose of GFP (green fluorescent protein)?

A

Using GFP with fluorescence microscopy allows for live imaging to be performed and track movement within cell of organism

55
Q

Cell fixation

A

cells are locked in place on glass slide and have permeable membranes that allow large molecules to pass through

56
Q

What is immunofluorescence?

A

Use antibody with fluorescence dye to detect specific protein using fluorescence microscopy

57
Q

What is one purpose of using immunofluorescence to study proteins in cells?

A

to provide information about location and amount of protein in cell

58
Q

Confocal microscope

A

A specialized type of fluorescence microscope that builds 3D images with laser beam and computer
Collects different optical sections and reconstructs sections to create 3D images

59
Q

Advantage of confocal microscope

A

High resolution 3D images to view cellular structures in great detail

60
Q

Disadvantage of confocal microscope

A

More expensive than fluorescence microscope

61
Q

Electron microscope

A

uses a beam of electrons instead of light to achieve higher magnification

62
Q

Advantage of electron micrsocpy

A

Highest magnification and best resolution (no limit)
Easily visualize organelles within cell

63
Q

Disadvantage of electron microscopy

A

Specimens require many steps for preparation
Not possible to view living cells (no live imaging)
Very expensive

64
Q

What is the major difference between an electron microscope and light/fluorescence microscopes?

A

Can see organelles in greater detail since diffraction limit does not apply since electron microscope is not using light

65
Q

Transmission electron microscope (TEM)

A

Requires thin sectioning and coating with heavy metals to specimen

Able to view structures as small as 1 nm

66
Q

Scanning electron microscope (SEM)

A

Generates 3D image, but only the surface of structure can be visualized

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Cell Biology (7 decks)

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