Lab Test Flashcards
What is Artemia?
A tiny extremophilic arthropod
What is diapause?
A dormant state characterized by a very low metabolic rate
What life stage is diapause present in?
Cyst
What happens when environmental cues resume normal development?
Cells begin to metabolize and divide, and nauplii emerge from the cysts
How long does it take for nauplii to emerge from a cyst once normal development has resumed?
16 hours
What must occur in order for the nauplii to grow in adults?
Multiple molts
What allows the cysts to survive diapause?
Heat shock proteins
What are heat shock proteins?
Proteins that associate with other soluble proteins and help prevent their denaturation
What other functions can HSPs have?
Function to monitor general protein activities and chaperone new or partially denatured proteins to fold into their correct shape
What is the function of HSP90?
Necessary for chaperoning normal protein synthesis and folding
What is the function of HSP70?
Chaperone protein folding, and important in cold survival during diapause
What is the function of p26?
Small heat shock protein mainly found in cysts
What is the function of ArHSP22?
Prevents irreversible stress induced by protein denaturation
What is the function of HSP21?
Contributes to stress tolerance during diapause
What are the three types of micropipette and how do they differ from one another?
P20, P200, P1000. They are each optimized to a different volume
Describe how to use a micropipette
Set it to the right volume, put a disposable tip on the end, push the plunger down to the first stop, put it in the liquid, slowly release, do not touch the walls of the container, hold the tube at eye level, press the plunger to the second stop to release the liquid, press the ejector button to dispose of the tip
What color are the micropipette tips?
P20,200 = yellow, clear. P1000 = blue
What do the numbers on the P20 mean?
Black = 1-20, red = tenths place
Should you wear gloves when working with a micropipette?
no
What does Bradford reagent do ?
Binds proteins in an acidic medium causing a color change from brown to blue
How is protein concentration measured in this lab?
By using Bradford reagents and measuring the absorbance of blue using a spectrophotometer
What is used as a standard to compare unknown samples to (in the spectrophotometer)?
BSA ( bovine serum albumin)
What is BSA?
A protein found in cow blood, very water soluble and stable
Why does the BSA need to be diluted?
Because the data is standardized between 0.20 and 1.00 and the sample conc is 1.4
What is the absorbance on the spectrophotometer?
595 nm
What must you use to wash the Bradford/protein off of the cuvettes?
Methanol
What is a blank?
A cuvette of the solvent used to dilute the material you want to know the absorbance of. Used in order to ensure that the spectrophotometer is only reading the absorbance of the reaction
Describe the process of using a spectrophotometer
Turn it on, set wavelength to 595, make sure the sample compartment is ready, adjust display to 0% transmittance,place a clean empty tube inside, press mode key to change display to absorbance, check to see if light is flashing. Turn control knob until flashing stops, wipe blank with kim wipe and insert. zero instrument, repeat with other tubes.
Why do you need to construct a standard curve?
To determine the protein conc in artemia samples from known values
What is used to dilute BSA and Artemia?
PIPES buffer
Why is a protein assay performed?
To determine the conc of protein in each airtime life stage sample
Why is electrophoresis performed?
To separate proteins by their molecular weight
What kind of gel are proteins electrophoreses on?
Polyacrylamide
What kind of gel are DNA electrophoreses on?
Agarose
How are protein electrophoreses visualized?
Coomassie blue
How are DNA electrophoreses visualized?
UV Light
What does SDS do?
Disrupts the secondary structure of molecules so the protein denatures and becomes rod shaped. Also coats polypeptides with negative charges
Why do small polypeptides move further down the gel?
Because they can fit through the membrane pores whereas larger proteins cannot
Why are the samples boiled before electrophoreses?
To ensure that they have been denatured
What else is done to denature the proteins before electrophoresis?
They are treated with a reducing agent that breaks disulfide bonds within the proteins (DTT, Mercaptoethanol)
What technique is used for determining the molecular weight of a protein?
Electrophoresis and standard curving
What molecular weight marker is used in this lab?
Pink Plus Pertained Protein Ladder
How many proteins are in PPPPL?
1 proteins with molecular weights ranging from 175 - 10.5
How do you know when to shut off the electrophoresis?
When the blue dye reaches the bottom of the gel
What must you do before loading the samples into the gel?
Heat and centrifuge them
What causes the sample to sink to the bottom of the gel
Glycerol in the sample buffer
What is the sample buffer made of?
DTT, Glycerol, SDS, Mercaptoethanol, Tracking Dye
How do you transfer the proteins on the gel to a nitrocellulose membrane?
Setting up a stack in a blot module, that is inserted in a blotting unit. Current passes through the gel and moves neg. charged proteins out of the gel and onto the nitrocellulose membrane
What are the things required for nitrocellulose transfer?
Blotting pad, nitrocellulose, gel, filter paper, transfer buffer
Describe the staining process?
Place half gel in container of coomassie blue, set on shaker table, poor coomassie blue back in the bottle, rinse the gel with water, fill container with destaining fluic
What is TBS tween and what does it do?
Poured on the nitrocellulose, it is a buffer that protects the proteins
What is Western Blotting?
A technique that uses antibodies to probe for specific proteins on the nitrocellulose membrane allowing you to determine which samples contain the protein
What organisms are commonly used to grow antibodies?
Goat, rabbit, mouse
What structure do IgG antibodies commonly have?
Y
How are polyclonal antibodies produced?
By injecting the animal host with a purified antigen. The host produces a mixture of antibodies that are each specific to a different epitome of the antigen
How are monoclonal antibodies produced?
Inject a mouse with an antigen triggering an immune response, removing an antibody producing cell and cloning it in cell culture. One specific antibody for one epitome is produced.
What is the advantage of monoclonal antibodies?
They are very specific and less likely to bind to the wrong antigen
What are secondary antibodies?
Antibodies that bind to primary antibodies
What kind of host must a secondary antibody be made in?
A different host than that of the primary antibody. Must be made against the constant domain of the primary antibody as well.
What kind of enzyme is usually conjugated to the secondary antibody for Western Blotting?
HRP, an enzyme that catalyzes a light producing reagent
What are secondary antibodies often conjugated to?
A reporter molecule like an enzyme, dye or fluorochrome
What is chemiluminescence?
Emission of light due to a chemical process
What does HRP stand for?
Horse Radish Peroxidase
What does HRP do?
Catalyzes oxidation of luminal causing a release of light
What does the milk do to the nitrocellulose membrane?
Coats the nitrocellulose in casein (milk protein) to minimize nonspecific binding of primary antibody to the nitrocellulose
What do you use to wash the nitrocellulose?
TBS tween
Describe the Chemiluminescence procedure
Mix OR and luminal. Place nitrocellulose in plastic wrap. Pipette OR/luminol onto membrane. Place blot in cassette and go to a dark room. Lay a piece of autoradiography film on it. Remove the film and put it in the developer. Rinse the film in dH2O and the put it in the fixer. Hang the film to dry.
Why should you pour destain solution on the gel when you put the gel on the light box?
To keep it from drying up
What is the total magnification = to?
Mag of ocular x mag of objective (10x10 or 10x40)
What is the quality of the image based on?
The resolution attained by the microscope
What is the limit of resolution?
The minimum distance between two points that allows them to be seen as separate
How do you set up a microscope for Kohler Illumination
Plug in microscope, set turret to 0, put a contrast slide in, use 10x objective to focus on image, close field iris and focus the light by moving the substage condenser mount, focus the ring of light with the entering screws, open the field iris
What is an ocular micrometer?
The ruler inside the eye piece
How many micrometers is one little division at 10 and 40x objective?
10 = 10, 40 = 2.5
What does HEK stand for?
Human embryonic kidney
What is an immortalized cell line?
Any cell that has the ability to divide indefinitely
What are adherent and suspended cells?
Adherent: stick to substrate, suspended: float around
How do you tell if one HEK cell is healthier than another?
More healthy, spreads out more
Are HEK suspended or adherent?
Adherent
What is an LAI?
Laboratory Acquired Infection
What are the classification of risk groups?
1,2,3,4 (indiv and community)
What risk group are HEK?
2
What does containment level mean
The minimum physical containment and practices required for materials and toxin handling in the lab
What is a BSC?
Biological safety cabinet, has an air curtain to prevent unfiltered air from entering
How can the laminar flow be interrupted?
Hand movement up/down or in/out and having too many items inside
What kind of filter is used?
HEPA
What is used for sterilization?
UV light and 70% ethanol
