Review 1 NOT COMPLETE

Which kind of tissue is shown?

Blood

Which kind of tissue is shown?

Liver

Which kind of tissue is shown?

Cheek

Which kind of tissue is shown?

Skin

Which kind of tissue is shown?

Neuron

What are the different types of microscopes and their characteristic features?

• Fluorescence Microscopy

  • Visualization of target proteins via fluorophores

• Electron Microscopy

  • significantly higher resolution, high zoom, NO LIVE SAMPLE SINCE ITS IN A VACCUMM

• Electron Cryo-microscopy

  • Allows for visualization of PROTEIN COMPLEXES, near atomic resolution

• Super-resolution Microscopy

  • allows for visualization of molecular interactions

• Confocal Microscopy

  • Uses PINHOLES to illuminate points of interest. HIGHER RESOLUTION AND CONTRAST THAN WIDEFIELD

• Stochastic Optical Reconstruction

  • uses sets of fluoresence chemically tagged areas SWITCHED ON AND OFF, reproducing high 3D images

What is limit of resolution?

SPECIFIES RESOLVING POWER

LOR is the smallest distance by which two neighboring points can be separated and be observed as separate entities

What is resolving power?

This is the ABILITY of a microscope to distinguish two closely spaced points

What is Abbe’s Equation?

Used to calculate the LOR

l.r. = 0.61x wavelength / numerical aperture

As N.A. increases, resolving power increases

What is working distance?

This is the distance between the specimen and the objective lens

What is the formula for total magnification?

Eyepiece magnification x objective magnification

How do you calculate the average length of a cell using the magnification of microscope?

Field of View (FOV) / number of cells present in middle line

What is cell lysis?

This is the breaking open of a cell to release its contents

Can be broken open via mechanical methods, chemical, or sound waves

What is a lysate?

The LYSATE refers to the mixture of the cell components after being broken open

What is homogenization?

Homogenization is the breaking down of tissue to a single uniform substance TO BE THEN LYSED

What is a homogenate?

This is the result of breaking down tissue to a uniform solution

What are the steps to be followed for centrifugation?

1. Check the cap to make sure it is closed

2. Balance the centrifuge by placing the tubes opposite each other or substituting with a tube of equal mass

3. Set to RPM or G

4. Make sure the container is closed

What is cell fractionation?

This is the breaking open of cells and then separating based on SIZE AND DENSITY using CENTRIFUGATION

What is the supernatant?

After centrifugation, this is the liquid solution that was not pelleted

What is the purity of cell fractionation?

This refers to the purity of the cell component separated

The purity of the fraction collected

What is differential centrifugation?

This is the concept of different cell components being separated at different centrifugation speeds

How are the different cell components separated based on size and density with varying centrifugation speeds?

Different cell components must be separated one at a time

• Low speed

  • Large dense cell components

  • Nuclei

  • Unbroken cells

• Medium speed

  • Mitochondria

  • Lysosomes

  • Peroxisomes

• High speed

  • MICROSOMES

  • golgi fragments

  • membrane vesicles

• Very high speed

  • Ribosomes

  • Viruses

  • Proteasomes

What is SDS-PAGE

Sodium dodecyl sulfate - Polyacrylamide Gel Electrophoresis

Denatures proteins and gives them a uniform NEGATIVE charge to separate them BASED ON SIZE ALONE

What is the function of SDS

SDS is a DETERGENT that denatures proteins

Important for linearizing the proteins and assigning a negative charge

What is beta marceptoethanol used for?

Used in the SDS PAGE process

It is used to BREAK DISULFIDE BONDS S-S, helps in denaturing the proteins

What is bromophenol blue used for in the SDS PAGE process

It is usually a component of the LYSIS BUFFER

It is used to allow for the tracking of the proteins as they move through the gel by color

What is glycrol used for in the SDS PAGE process?

ADDS WEIGHT so that the protein sample can enter the gel well

Prevents the sample from dissolving

FOUND IN LYSIS BUFFER

What are the components of the Lysis Buffer

• SDS

• Glycerol

• bromophenol blue

• pH buffer

• mercaptoethanol

What is the assembly of the blot stack during transfer

SDS PAGE gel is transferred to a MEMBRANE

1. Pad (either - or + depend on where the gel and membrane are) +

2. Filter paper

3. Membrane

4. Gel

5. Filter paper

6. Pad (-)

Remember the gel has (-) proteins so it must go from - to positive

What are the steps of western blot?

1. Perform electrophoresis to get gel

2. transfer gel to nitrocellulose membrane

3. Blocking buffer (Skim milk)

4. Incubate with primary antibody

5. wash buffer

6. incubate with secondary antibody

7. wash buffer

8. detect proteins

What is the function of western blot?

A Western blot tells us if our sample contains our target protein

ABUNDANCE OF THE PROTEIN AND SIZE OF THE PROTEIN

How do we prepare our samples for SDS-PAGE?

1. Extract target tissue

2. Add lysis buffer (opens the cells)

3. Add LAEMMLI BUFFER (contains the SDS, glycerol, mercaptoethanol, buffer, and bromophenol blue)

4. Heat

What are the types of buffer components for SDS PAGE and blot transfer?

• Sample buffer (LAEMMLI BUFFER)

• Running buffer

  • fills the gel tanks for SDS PAGE, allows proteins to migrate down

• Gel Buffers

  • Tris HCl and SDS

• Blocking buffer

  • DRY SKIM MILK

  • CASEIN-^

• Wash Buffer

In SDS, what does the location of the protein band tell you?

The lower the band is = THE LIGHTER THE PROTEIN IS

What is a plasmid?

This is EXTRACHROMOSOMAL DNA that is circular and contains genetic information such as antibiotic resistance

What are restriction enzymes?

These are enzymes that cleave specific RESTRICTION SITES, yielding different pieces of DNA which can then be TRANSFORMED into a cell and attached to a plasmid

What is cloning?

This is the process of generating multiple copies of GENETICALLY IDENTICAL copies of DNA, cells, or organisms

What is a bacterial library, and how do we make genomic and cDNA libraries?

A bacterial library refers to a culture of bacteria, each with a plasmid containing a target gene

• Genomic library

  • Many bacteria, each containing different parts of the human genome in their plasmid

• cDNA library

  • Represents only the EXPRESSED genes

  • mRNA code only

What is Hydrophobic Interaction Chromatography?

HIC utilizes high salt conditions to bind the hydrophobic parts of a protein SUCH AS GFP

• High salt = takes the water away resulting in hyrophobic regions being exposed, BINDING TO COLUMN

• Low salt = attached protein is eluted out

What are the different buffers used in HIC

Binding buffer = HIGH SALT

Elution buffer = LOW SALT

What are the different stages of purification during HIC?

1. Equilibration - high salt, prepares the column

2. Binding buffer - very high salt, exposes nonpolar and causes binding

3. Wash - medium to high salt, washes weakly nonpolar CONTAMINANTS

4. Elution - Low salt, releases the hydrophobic protein

Role of antibiotic resistance gene such Ampicilin resistance gene in plasmid and their impact on
transformed bacterial growth

Antibiotic resistance gene in a plasmid ensures that only bacteria that have taken up the plasmid can reproduce

Therefore only the bacteria that have successfully transformed the plasmid can continue to grow

Role of inducer such as arabinose in lab engineered GFP bacteria

In GFP bacteria, arabinose is needed to bind to the GFP regulatory protein

HELPS TURN ON EXPRESSION

role of lysozyme

The lysozyme is used to break open cells, releasing its contents which can then be centrifuged to isolate the proteins in the supernatant

Quorum sensing, and their role in bacterial growth, survival and how to identify the different mutant strains and wild type

Quorum sensing is used in bacteria as cell to cell communication

They release and detect signaling molecules which help the bacterial colony identify when its reached critical growth