protein visualization

Question 1

what is an isotope?

Answer 1

an istope is atoms with same number of protons and different number of neutrons

Question 2

what are radioactive isotopes?

Answer 2

radioactive isotopes are atoms with different number of protons and neutrons making it highly unstable, they will break to become stable and will release an electromagnetic radiation

Question 3

where are radioactive isotopes found?

Answer 3

their found throughout the periodic table and you can buy molecules with radioactive isotopes attached to them

Question 4

what is the radiation radioactive atoms release during decay?

Answer 4

beta particles

Question 5

how can we use beta particles to see the exact location of radioactive atoms in a speciemen

what is this method called?

Answer 5

when a silver halide is placed over a radioactive speciemen , the silver halide will become a metallic halide and appear as black dots on the electron microscope

it is called autoradiography

Question 6

If we saw the H3 urdine puffs(black dots) in an insect what can we conclude?

Answer 6

since urdine is used in transcription , areas with lots of black dots concludes high transcription levels

Question 7

what is the pulse chase experiment

Answer 7

a form of autoradiography to track the movement of secretory proteins in the endomembrane system

pulse= proteins are introduced to radioactive amino acids (will become tagged)

chase= newly made proteins will no longer be introduced to the radioactive amino acids and are now untagged , we will now just take pictures of the movement of proteins after periods of time

Question 8

what do we have to do to proteins in order to visualize them?

Answer 8

in order for us to see proteins we have to probe them with something

Question 9

what are the two effective techniques we use antibodies for

Answer 9

1)antibody linked to fluorescent dye(immunofluorescence , and uses a fluorescent microscope)

2) immunogold antibody(gold particles and uses immungold microscopy)

Question 10

what proteins are best used for visualization?

Answer 10

antibodies ,

Question 11

what makes up the antibody structure

Answer 11

1)2 light chains and 2 heavy chains
2)two identical binding sites

Question 12

what is specific to an antibody ?

Answer 12

an antigen is specific to the antibody , it will bind tightly to the antibody

the antigen is so specific that it would notice a single amino acid difference in the peptide chains

Question 13

what do our immune antibodies do ?

Answer 13

they recoginze and destroy foreign molecules, invading bacteria and viruses

Question 14

what are antibodies made of ?

Answer 14

antibodies are made of a specific class of white blood cells known as B Lymphocytes also known as B cells

each B cell membrane has receptors on them that are receptors for specific antigens

Question 15

what happens when the antigen binds to the antibody on the b cell membrane?

Answer 15

the B cell is stimulated to divide and it will produce a lot of antibodies in soluble form

Question 16

how can we get stronger flourescence signal?

Answer 16

we can get a stronger signal by using two antibodies together where only the second one will have the dye

Question 17

how can we produce antibodies ?

Answer 17

1)take an antigen (from human) and inject into mouse (b cells will produce antibodies for that antigen )

2)take the primary antibodies made by the rat and insert it into a turtle and the turtle will produce secondary antibodies for that antigen

Question 18

what is a fluorescent molecule?

Answer 18

it absorbs light at a specific wavelength and emits a light at a different longer wavelength

Question 19

what are the three synthetic fluorophores or fluorochromes?

Answer 19

1)fluoroscien= produces a green colour
2)rhodamine=red colour
3)DAPI= produces a blue colour and since its dna it wont need a antibody

Question 20

what does a fluorescence microscope contain?

Answer 20

1)filters at key points that block the unwanted wavelengths of light( such as the first barrier allows only blue at 450-490 and second barrier allows only fluoresceine between 520-560)

2) beam splitting mirror= reflects a lift to the speciemen and transmits an even longer light from the speciemen ( reflects below 510 and transmits above 510)

Question 21

what is the immunogold antibody ?

Answer 21

-antibody is attached to a gold particle
-viewed under an EM where itll appear as black dots and the gold particles are electron dense

Question 22

what is the GFP?

what does it allow for

Answer 22

a GFP is a protein extracted from jellyfishes that glows under blue light

it allows for real time fluorescence micrope observations of living species movement

Question 23

why does the GFP fluoresces ?

Answer 23

due to the positioning of three R groups in the middle of the beta barrel (form a fluorophore)

Question 24

what is a chimera?

does binding of a gfp to a different protein affect the protein?

Answer 24

the GFP plus a different protein is known as a chimera
the gfp will have little to no affect on the protein

Question 25

where does the gfp gene have to be inserted?

Answer 25

it has to be added to the begining or end of the coding region of the gene of interest this way the protein will now be transcribed and translated with the gfp and will now glow

Question 26

how have scientist altered the GFP colours?

Answer 26

they have created different colours by changing the gfo sequence and the structure in zebrafishes each protein is made by dna that would glow when in contact to certain fluorescent dyes

Question 27

what is resolution?

Answer 27

it is the ability to see two objects that are very close together more clearly

Question 28

what is the relationship between resolution and distance between the two particles? | what are the components of d

Answer 28

the resolution is equal to the distance the distance has three parts A= the half angle of the cone of light that shines from spicemen to lense N = reflective index medium that is between the obj lense and the speciemen w=which is the wavelength of the light used

Question 29

the longer the wavelength the greater the resolution ? T/F

Answer 29

FALSE , the shorter the wavelength the greater the reso

Question 30

what does the EM use?

Answer 30

the EM uses an electron beam to pass throught the speciemen , shorter the electron beam the greater resolving power

Question 31

what are the two EM?

Answer 31

TEM= produces an image based off the transmition of electrons through the speciemen( 0.3-0.5) SEM= produces an image based off the electrons bouncing off of the speciemen , large with no internal organelles and has a wavelenghth of 5-10nm

Question 32

how can you focuses the negatively charged electron beam ?

Answer 32

via electromagenetic lenses found in the wall of the column

Question 33

what is the thin speciemen held by ?

Answer 33

it is supported by a small metal grid that has been inserted with tweezers to a grid holder

Question 34

what are the functions of the different parts of the EM?

Answer 34

1)top column= has cathedor made of tungeston wire filaments that heat up to produce electrons 2) the condenser electromagnetic lenses focus on the electron beam to the speciemen 3) the rest of the lenses project the electron beams further down into the speciemens 4) electrons then pass down into the photographic plate

Question 35

the fewer electrons that hit the plate the more dark spots form on the image, why?

Answer 35

1)as electron beam passes through speciemen , it hits some speciemen atoms and it will move it off its track 2)the greater the speciemen atoms the greater the diversion of electrons 3)when diversion of is enough then the electrons dont hit the plate and are removed and will now produce dark spots indentifying the speciemen had high density

Question 36

why do we need to pump air during the EM?

Answer 36

in order to create a vaccum, because if u dont create a vaccum the electrons will scatter due to gas particles

Question 37

why do we use stains in EM?

Answer 37

common atoms such as H or C wont scatter the electrons properly hence why the heavy metal dye osmium is used and helps scatter the electrons properly

Question 38

how do we do immunogold microscopy?

Answer 38

we use the tem where the gold particles will appear as dark spots

Question 39

what does the SEM show?

Answer 39

SEM shows the shape and surface of cells without water. Cells are carefully dried, coated with gold, and then viewed with an electron beam. Example: T4 bacteriophage at 275,000× magnification.