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A way to look at tissue without using chemicals?
- must be quick--> use liquid Nitrogen (-196 degrees Celsius) to prevent ice crystals
How do you fracture your frozen specimen?
How will the specimen look?
- strike it (must be in a chamber at low temperature)
- fragments that look like glass
Where does the specimen fragment?
lines of weakness; in the cell membrane, its the point in the middle of the lipid bilayer
What happens to protein during freeze fracture?
they dont get cut; it goes around them
After fracturing, what happens?
- you put it in a vacuum to get rid of surface ice (sublime)= this is the etching part
- Then, you put heavy metal (at an angle) and carbon (put on straight; uniform layer)
- get rid of tissue
Why put on heavy metal sideways?
it causes a shadow; to produce variation in the color, thickness of the metal matters
What would be the appearances of surfaces A and B produced by freeze fracturing according to the DD, RUM, and FMM models?
A= outer surface
B= inner surface
- DD: the outer would be rough; inner smooth
- RUM: both smooth
- FMM: both rough, but inside more so
Why is carbon applied?
to stabilize the structure
Fluid Mosaic Model: why are membranes fluid?
becuase most of the molecules aren't rigidly fixed in place
How do we know the membrane is fluid?
becuase you can generate antibodies, attach dye, and see what happens to the color
- take different types of cells and label proteins on the surface of those cells
- ex: mouse cell with a particular protein stained and a human protein stained differently= try to get fusion
- Once fused, the initial look would be half-half of each stain
- However, once time passes, it is no longer half and half. The colors spread out
True or False:
Proteins MUST be labeled before fusion.
False: no they do not have to
Explain a proper analogy for the proteins in a membrane.
- proteins are like icebergs floating in a sea of lipid
- If that's the case, stopping lipid movement can freeze proteins in place
How do we stop lipid movement?
- add cholesterol
- freeze the lipids by lowering the temperature
If you stop lipids, what should happen?
it should stop proteins
If you take any lipid and its liquid and you start lowering the temperature, what?
you shoudl reach a temperature where the liquid is converted to solid= transition temperature
Experiment two pertaining to the fluidity of a membrane?
- cell with labeled proteins
- take a laser and get rid of dye in a particular spot, called photobleaching; eventually, that spot will fill up again
Above the transition temp, it is __. Below, it is__.
As you __, mobility of proteiins __.
- lower temperature
- stops or becomes slower
What does fluidity depend on?
on lipids and how easily they can pack together
If they pack together easily, what?
If it is difficult?
- you have a higher transition temperature (don't have to lower as much)
- you have a lower transition temperature becuase you really have to lower the temperature to see a change
If talking about phospholipids, the transition temperature will depend on?
their ability to pack together
What features affect fluidity? How
- double bonds (kinks)= cause difficulty during packing togeher and bring the transition temperature down
- length of fatty acid chains: if long, packing together is easier/ if smaller, its harder because they move faster
Why is it easier for a longer fatty chain to pack together?
Transition temperature does what with what?
- if long, increasing surface area for interaction
- transition temperature increases with increasing chain length
The longer the chain, the __
higher the transition temperature is
Of the two features that affect fluidity, which is the most important?
unsaturated and saturation (double bonds)
If you have a whole bunch of lipids in different beakers, the transition temperature depends on the type of lipid. It'll range from 0-60 degrees Celsius.
Explain if the lipids are pure and if they are different.
- pure: transition temp is a sharp value; only have to go a couple of degrees down to get transition
- in membranes, its a raw value due to mixture; no real sharp transition temperature--> Broad Range
- wont get it down in a couple of degrees
What characteristics would you expect the fatty acid portion of membrane phospholippids to have in bacteria living in the Arctic Ocean?
- lots of double bonds and shorter chains
- you want a lower temperature for transition temp