Lecture 3 and 4: Cell Biology Techniques, Part I and II Flashcards
What is whole cell lysate?
collection of cells produces cell lysate which is single cell suspension of desired cells
How does whole cell lysate become cell lysate?
Through lysis buffer (or any detergent)
What is cell lysate/homogenate/extract?
Sample after adding lysis buffer for plasma membranes disrupted and sample has all cellular components
What is cell fractionation?
separate cellular components from one another and isolate organelles via centrifuge
Cell fractionation is not used to ______ but to _______
isolate specific proteins; segregate different cellular components from a cell lysate
Second higher speed centrifugation:
Pellet contains mitochondria, cytosol, and other organelles
Cell fractionation use centrifuge to spin at _____ to pul down heavier molecules
different speeds
First low speed centrifugation results:
Pellet contains nuclei
Supernatant contains cytosolic proteins and organelles
The purpose of cell fractionation is to
evaluate 3 types of proteins and reduce complexity of sample
3 types of proteins resulted in cell fractionation:
- Cell lysate: all proteins
- Nuclear proteins: proteins found only in nucleus
- Cytosolic proteins: all proteins outside of nucleus
What is SDS-Page?
Separates protein in a mixture by size and allows to analyze proteins in cell lysate or cell fractionation samples
SDS Page contains:
SDS: detergent that denatures proteins and coats proteins with negative charge
Beta-mercaptoethanol (BME): reducing agent that eliminates disulfide bridges
Why is negative charge by SDS important in gel electrophoresis?
negative charge pulls down protein and allow for all proteins to have uniform charge
After SDS-PAGE, what are the options to analyze?
- Coomassie blue
- Western blot
What is coomassie blue?
Stain to detect and visualize all proteins in gel as stain binds to basic amino acids
What is the purpose of coomassie blue?
- ensure cell lysates from different samples have equal protein loading
- determine if protein was successfully purified
How is Western blot technique performed?
- Use SDS Page gel to separate protein mixture in sample
- Transfer separated proteins from gel to membrane
- Primary antibody recognizes specific protein on membrane
- Secondary antibody binds to primary antibody and used for visualization, more signal on membrane indicates more protein in cell
Purpose of Western blot
Measuring amount of specific protein with antibodies
How are Coomassie blue and Western blot similar and different
Similiar: both analyze proteins after SDS PAGE
Differ: comasie is nonspecific stain while western blot is specific
What are antibodies useful for?
Useful as they recognize bacteria, viruses, and other foreign molecules that cause harm
What is protein structure of antibodies?
- 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)
Does an antibody have a binding site?
Has 2 binding sites (Vh and Vc) which can bind to an antigen
What is an antigen?
substance that stimulates immune response
How are antibodies naturally made?
- 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
What happens to secreted antibodies?
Bind to antigens and target them for destruction by immune cells
What are polyclonal antibodies
mixture of antibodies that bind to different places on same antigen
Process of making polyclonal antibody
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
What is the purpose of an immunoprecipitation (IP) ?
uses antibodies to purify protein complexes and assess protein-protein interactions
Why are monoclonal antibodies ideal for drug treatment and scientific research?
- Purified from hybrid cells which provide infinite supply of antibodies
- Have low variability between batches to be ideal for research and drug treatment
Process of making monoclonal antibodies
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
How is an immunoprecipitation performed?
- 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
What is protein purification?
purifying thousands of proteins from cell lysate into one protein
What is the trick of process of protein purification?
Giving protein of interest a handle bar with fusion protein
Fusion protein
Genetically engineered protein that is used to isolate a specific protein
Can contain a tag
GST Tag
GST protein attached to gene sequence to make fusion protein which is inserted into cells
What is taken advantage of GST for protein purification?
GST bind to GSH with very high affinity
Steps on GST tag used to purify specific protein from a cell lysate
- Create cell lysate containing mixture of cells including cells expressing fusion proteins
- 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 - 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
How is GST able to bind with glutathione after binding with GSH?
GST and GSH bind is not permanent due to being weak non covalent interaction
How can coomassie blue be used for protein purificaiton?
Used to determine which sample has purified protein after purification
How should 5 samples look like with coomassie blue after protein purification?
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
Light microscope
Uses light source to view single cells and internal structure of a cell
3 requirements for light microscope
- Light source must be focused on specimen
- Specimen must be transparent
- Set of lenses (objective and eyepiece) must focus the image
Advantages of using light microscope
Easy and cheap to use
Able to do live imaging and view internal structures
Disadvantages of using light microscope
Specimen must be transparent and colorless
Internal structures are difficult to see
Limit to magnification since light source is used
Fluorescence Microscope
Use light source, but structures can be seen in greater detail with fluorescent labels
Each fluorescence label has….
excitation and emission wavelength
How does a fluorescent label work?
- First filter selects excitation wavelength that will excite label in specimen
- When wavelength reaches label, it will emit light at different wavelength and label comes back to ground state which releases photon
- Second filter selects wavelength emitted by label so photon can be detected, cause visualization while rest of specimen is black
Advantage of fluorescence microscopy
Live imaging can be performed
3D structures by confocal
Disadvantage of fluorescence microscopy
More expensive than light microscopy
Fluorescence signal will fade over time (photobleaching
Short wavelength =
high energy
Long wavelength =
low energy
How do scientists attach fluorescent tags to specific proteins?
Fluorescence tags can be added to DNA sequence to create fluorescent protein (aka fusion protein)
What is a common fluorescent tag attached to proteins?
GFP (green fluorescent protein)
What is the purpose of GFP (green fluorescent protein)?
Using GFP with fluorescence microscopy allows for live imaging to be performed and track movement within cell of organism
Cell fixation
cells are locked in place on glass slide and have permeable membranes that allow large molecules to pass through
What is immunofluorescence?
Use antibody with fluorescence dye to detect specific protein using fluorescence microscopy
What is one purpose of using immunofluorescence to study proteins in cells?
to provide information about location and amount of protein in cell
Confocal microscope
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
Advantage of confocal microscope
High resolution 3D images to view cellular structures in great detail
Disadvantage of confocal microscope
More expensive than fluorescence microscope
Electron microscope
uses a beam of electrons instead of light to achieve higher magnification
Advantage of electron micrsocpy
Highest magnification and best resolution (no limit)
Easily visualize organelles within cell
Disadvantage of electron microscopy
Specimens require many steps for preparation
Not possible to view living cells (no live imaging)
Very expensive
What is the major difference between an electron microscope and light/fluorescence microscopes?
Can see organelles in greater detail since diffraction limit does not apply since electron microscope is not using light
Transmission electron microscope (TEM)
Requires thin sectioning and coating with heavy metals to specimen
Able to view structures as small as 1 nm
Scanning electron microscope (SEM)
Generates 3D image, but only the surface of structure can be visualized
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