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Eukaryotic cellular biology 1 > Techniques > Flashcards

Techniques Flashcards

1
Q

How does a light microscope work?

A

Light from a lamp passes through a very thin specimen and is focused and magnified through a series of lenses

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2
Q

Why is there a limit to the magnification a light microscope can get?

A

Light microscopes use visible light, and at a point the wavelength can’t get any shorter so we can’t see it

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3
Q

What is the order of the wavelengths in the electromagnetic spectrum from shortest wavelength to longest wavelength?

A

Gamma rays -> x-rays -> UV light -> visible light -> Infrared light -> microwaves -> radio waves

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4
Q

Which has higher energy? Blue light or red light?

A

Blue, shorter wavelength

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5
Q

How to wavelengths of light affect how a specimen is viewed?

A

A specimen is seen based on how it interferes with the wavelength of light passing through it. It is more likely to interfere with a shorter wavelength then a longer one

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6
Q

What is resolution?

A

How far apart two objects need to be in order to be seen as separate objects

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7
Q

What are the 4 kinds of light microscopy? What do they allow us to see?

A

Brightfield: what we usually do
Phase contrast: can see if cells are alive
Differential interference contrast (DIC): gives an image that is more 3D and gives a better idea of structure
Fluorescence microscopy: makes targeted proteins or molecules glow

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8
Q

What are the 2 kinds of electron microscopy? How do they work?

A

Scanning electron microscopy: the sample is coated with an electron dense material (gold) and electrons are bounced off to create a 3D image
Transmission electron microscopy: a very thin sample has a beam of electrons passed through it and produces an image

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9
Q

What is a microtome?

A

A deli slicer for cells that slices a specimen into very thing slices

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10
Q

Why do cells need to be fixed and permeabilized? How is it done?

A

Proteins and structures need to fixed in place to make the cell look as though it is alive. It needs to be permeabilized to get the dye inside. Cells are fixed by using chemicals (such as formaldehyde), permeabilization is done with mild detergent to poke a few holes in the membrane

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11
Q

What is DAPI?

A

A fluorescent blue dye that binds to DNA

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12
Q

What are vital dyes?

A

Dyes that penetrate cells and are seen under brightfield light microscopy

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13
Q

What are the two stains used in HME staining? What do they stain?

A

Hematoxylin and eosin. Eosin stains DNA a dark purple. Hematoxylin stains all proteins pink

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14
Q

What is fluorescence? What causes things to fluoresce?

A

Fluorescence is the absorption and emittance of light. When light is absorbed, electrons become excited and jump to a higher orbital, then fall back down and emit light

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15
Q

Is the wavelength absorbed and emitted the same in fluorescence?

A

No. Emitted light is a longer wavelength

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16
Q

How does fluorescence microscopy work?

A

Light of all wavelengths is shined on the sample, so a barrier filter is used to stop all wavelengths but the excitation wavelength. A second barrier filter is used to stop the excitation wavelength and let through the one we want to see

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17
Q

What are antibodies? How are they structured?

A

Proteins produced by mammalian immune systems that attach to foreign antigens. They have a common region that is the same for every antibody from that species and a variable region that attaches to the antigen and is unique to every antibody

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18
Q

How are antibodies used in fluorescence microscopy?

A

A fluorescent probe is attached to an antibody, and the antibody will then bind to the antigen and fluoresce. It gives a very specific location of a protein of interest

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19
Q

Why do researchers use double labelling in fluorescence microscopy?

A

Visualizing one labelled antibody, then another, then overlapping the photos will allow researchers to determine if colocalization occurs

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20
Q

What are the advantages of antibodies?

A

They are very specific to one particular protein, and allow researchers to determine the expression and location of a particular protein in a cell

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21
Q

What are the disadvantages of using antibodies?

A

They take a lot of time and effort to make, and there’s no guarantee they’ll even work. You also need to kill the cells, so protein expression over time can’t be tracked

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22
Q

What is the difference between direct and indirect antibody use?

A

Direct is when the fluorescent probe is attached to the primary antibody that attaches to the antigen. Indirect has several labeled secondary antibodies attaching to the primary antibody

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23
Q

Why is it better to use secondary antibodies in fluorescence microscopy?

A

The signal is amplified, the same secondary antibody can bind to many different primary antibodies (which gets rid of the need to constantly relabel the primary antibody)

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24
Q

How can proteins be visualized while still alive?

A

Creating a fusion protein by adding the genetic sequence for GFP on the end of the genetic sequence for the protein of interest

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25
What can be a problem with using GFP?
Attaching this giant thing to the end of a protein might change its shape and function
26
What is hydropathy analysis?
A very simple graph that predicts the number of membrane spanning components in a protein
27
What does a hydropathy graph look like?
Has a line in the middle, a hydrophobic amino acid is above the line, a hydrophilic amino acid is below the line. Hydrophobic peaks indicate the protein likely crosses the membrane
28
What is a cell lysate?
A mixture of organelles, bits of membrane, and macromolecules in a solution
29
When would we want to keep organelles and proteins intact in a cell lysate?
If we wanted to separate the organelles through centrifugation
30
How would a cell lysate be prepared if we wanted to keep organelles and proteins intact?
Would disrupt the membrane by changing the osmotic pressure and causing the cells to swell up and pop. A homogenizer can also use gentle mechanical shearing to break the membranes. Can use mild, non-ionic detergents to break up the membrane
31
How do detergents work?
They are amphipathic molecules that intercalate into the membrane and make lipids and phosphates soluble
32
When would we want to tear everything apart in a cell lysate?
For immunoblotting
33
How would we prepare a cell lysate if we wanted to tear everything apart?
SDS would be added to tear apart the membrane and denature all proteins
34
How is gel electrophoresis done with proteins?
Proteins from a cell lysate are treated with SDS and a reducing agent (beta-mercapoethanol), and boiled to denature them. The SDS also coats all the proteins with a negative charge. The samples are loaded on the gel and an electrical current is run through the gel and the proteins migrate towards the positively charged electrode. Can then stain the gel with coomassie blue to see all the protein bands, or not stain it and do an immunoblot
35
After running the gel, how is an immunoblot done?
The proteins are transferred onto a nitrocellulose sheet. Primary antibodies for the protein of interest are added, and labeled secondary antibodies are added after. The secondary antibodies are labeled in a way that allows for location detection
36
What are loading controls used for? What is usually used as a loading control?
They are to show that equal amounts of sample were added in each lane, and that variations in band intensity are due to different levels of expression instead of different amounts of sample. Actin is often used because its always readily expressed
37
What is sub cellular fractionation?
Further breaking down a cell lysate
38
How does centrifugation work?
Under centrifugal force, substances will move at different rates based on size, shape, and density and also depends on the density and viscosity of the solution
39
What are the 2 types of centrifugation?
Fixed angle rotor and swinging bucket rotor
40
What are sedimentation coefficients?
The speed at which organelles separate based on size, shape, and density
41
What is transfection?
Adding a vector containing a gene for a protein of interest into a cell by poking a few holes in the membrane with lipids
42
Why don't mammalian cells express the proteins encoded by the transfected DNA very long?
Only very rarely is the plasmid integrated into the chromosomal DNA, so it doesn't get copied during replication. After cell division, which is about 24 hours, the daughters cells don't have the plasmid
43
What is the difference between transient transfection and stable transfection?
Transient is when the DNA stays as a plasmid, so it's temporary. Stable is when the plasmid gets integrated into a chromosome, but this is very rare and completely random
44
Why would we want to do transfection of an endogenous protein?
If we wanted the cell to make more of the protein and overexpress it
45
What are fusion proteins?
A combination of 2 different proteins created by adding the genetic sequence of something else on the end of the sequence of the protein pf interest
46
What is often added to a protein of interest to create a fusion protein?
GFP or an affinity tag/epitope
47
Why are epitopes often added to proteins?
Can add the antibody for the tag if we don't have the antibody for the protein of interest
48
Why would we want to get a cell to express less of a protein?
To determine if a protein is essential for a certain function
49
What do cells do naturally to knockdown expression of a protein?
RNA interference. It either blocks translation or causes the mRNA to be degraded
50
What are the steps in natural RNA interference?
A piece of RNA folds up into a hairpin, which the cell recognizes as abnormal and chops up into micro RNA with dicer enzymes. miRNA binds to the target mRNA through complementary base pairing and stop the mRNA from ending up as a protein
51
What is the synthetic version of the natural process of RNA interference?
siRNA or shRNA
52
What is the difference between siRNA and shRNA?
siRNA is synthetic miRNA that is transfected into the cell as synthetic double stranded siRNA. shRNA is made by transfecting the DNA encoding the shRNA into the cell. The cell will then express the shRNA, and it will fold into a hairpin and get degraded.
53
What is qPCR used for?
To track the amplification of a sequence in real time, and from the known amplification pattern, determine the amount of that sequence originally in the sample
54
What is a proximity ligation assay used for?
To determine if two proteins are interacting. Both proteins are marked with tags and will only fluoresce if they're close enough to each other
55
Why can't fluorescence microscopy or PLA guarantee that two proteins are interacting?
Fluorescence microscopy can tell us if two proteins are colocalizing, and PLA just tells us if they are really close together
56
What is a co-immunoprecipitation?
A specific protein is precipitated out of a cell lysate by using antibodies, and any other proteins binding to that protein will also come down with it
57
What are the steps for an immunoprecipitation?
1. add antibodies for the protein of interest to a cell lysate 2. leave for a bit to let the antibodies bind 3. Add protein A or G resin beads 4. centrifuge 5. Wash a few times to remove most of the impurities
58
Why is immunoprecipitation a separation and not a purification?
The solution can't be washed too many times, because protein interactions will get destroyed. There will always be some impurities, but the process separates out a protein of interest
59
Why isn't a loading control required for immunoprecipitation?
No claims about expression are being made, only if a binding partner is present or absent
60
Why would an immunoblot be done on the protein that was targeted for immunoprecipitation?
To prove that the immunoprecipitation was successful. If no binding partner was seen, this would prove that the experiment didn't screw up

Eukaryotic cellular biology 1 (14 decks)

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