PLBI Lab Quiz 3 - Lab 4 and 5

Question 1

genus and species name for arabidopsis

Answer 1

arabidopsis thaliana

Question 2

which family is arabidopsis from

Answer 2

brassicacae

Question 3

arabidopsis is related to which food groups

Answer 3

canola

Question 4

why is it ideal for plant biology research

Answer 4

small, fast growth, self pollinating, small genome

Question 5

what are transgenic arabidopsis lines

Answer 5

lines that have been genetically engineered to express a new trait not naturally found in them

Question 6

in arabidopsis, what is the foreign gene that modifies arabidopsis, and how is it integrated

Answer 6

the fluorescent protein marker, its it’s integrated into the genome so every cell in the transformed arabidopsis will express the fluorescent protein marker

Question 7

how is cell plasticity useful to us

Answer 7

allows regeneration of a whole plant from a single cell, so the single tranformed cell will be able to generate a whole plant that expresses the protein

Question 8

out of all these organelles, which ones express GFP vs RFP, and why are they labelled

Answer 8

mito, golgi, er, tubules, peroxisome - all GFP, so we can visualize their movemnts and morphology easily

Question 9

What do we use epifluorescence techniques to do in the lab (general)

Answer 9

to visualize fluorescent labelled structures within cells

Question 10

in this lab (4), what are we using epifluorescnece microscopy to visualize

Answer 10

cytoskeleton, organelles in root epidermal cell of arabidopsis

Question 11

name 2 challenges of autofluroescence

Answer 11

Background fluorescence in tissues (like leaf mesophyll cells) interferes with imaging due to chlorophyll and cell wall fluorescence.

Root epidermal cells are less autofluorescent, they’re more suitable for detailed imaging with less background interference.

Question 12

arabidopsis lines are genetically engineered to express flurorescence under which protein

Answer 12

GFP

Question 13

For the targeting mechanisms, organelles are labelled via [blank] or [blank]

Answer 13

targeting signals or binding proteins

Question 14

example of targeting signal and organelle

Answer 14

SKL peroxisome targeting sequence (for peroxisomes)

Question 15

example of binding protein and organelle

Answer 15

MAP 4 microtubule binding protein for microtubules

Question 16

high or low power brightfield is used to visualize cytoplasmic outer layer

Answer 16

low

Question 17

Why do more cells fluoresce in the transgenic Arabidopsis lines compared to bombarded onion cells?

Answer 17

foreign gene encoding GFP is stable in arabidopsis, but in onions, theres only a small localized region to express the gene so less fluroescence

Question 18

w

why is there more autoflurescnece in leaf tissue than root tissue

Answer 18

Leaf tissue contains chlorophyll, which has strong natural fluorescence under epifluorescence microscopy, leading to significant background signal.

Root cells lack chlorophyll, resulting in minimal autofluorescence and clearer imaging of labeled structures.

Question 19

Are the organelles in Arabidopsis root cells similar in morphology to those in onion leaf cells?

Answer 19

yes, similar morphology and comparable functions. but organization and distribution differ

Question 20

What does immunofluoresence microscopy do

Answer 20

visualize specific proteins, target them with antibodies conjugated to fluorescent proteins

Question 21

difference between epiflurosences and immuno in terms of localization

Answer 21

fluorescent = mislocalization, immunofluorescence = accurate localization proteins.

Question 22

lab 5 uses [blank] (direct or indirect) immunofluorescence, which involves [blank] antibodies to amplify the fluorescent signal for clearer imaging.

Answer 22

indirect, primary and secondary

Question 23

Primary Antibody

Answer 23

recognizes protein of interest, monospecific to reduce BG noise

Question 24

Secondary Antibody

Answer 24

conjugated to flurosecnet molecule, bind to primary antibody for enahnced visibility

Question 25

flurosecnet molecule in lab 5 use

Answer 25

alexa fluor 594

Question 26

lab 4 is about arabidopsis root epidermal cells whil lab 5 is about

Answer 26

immunofluroescent plasmodesmata in onion epidermal cells

Question 27

fixation process

Freshly sectioned onion epidermal peels are fixed in [blank] with paraformaldehyde and glutaraldehyde to preserve cell structure.

Answer 27

PBS

Question 28

enzymatic treatment

Cells are treated with [blank] to partially digest the cell wall, facilitating antibody penetration.

Answer 28

pectolyase and cellulase, digest cell wall for antibody penetration

Question 29

for blocking

Blocking buffer with [blank] reduces non-specific binding

Answer 29

BSA so it binds accurately

Question 30

Primary antibody (target protein of interest) incubation targets [blank], a component of [blank], specifically binding to callose around [blank] (the focus of lab 5…).

Answer 30

β-glucan, a component of callose, specifically binding to callose around plasmodesmata.

Question 31

Secondary antibody conjugated to [blank] (which fluorescent molecule) enhances fluorescence visibility.

Answer 31

alexa fluor 594

Question 32

Imaje J vs GIMP

Answer 32

basic adjustments like brightness, contrast, and overlays of multi-channel images.

pseudo-coloring and adding scale bars or insets for clearer publication-quality figures.

Question 33

Callose, a β-glucan polymer, forms around plasmodesmata, regulating [BLANK] transport.

Answer 33

intracellular

Question 34

double labelling capacity of immunofluorescence

Immunofluorescence allows for simultaneous visualization of [blank]

Answer 34

two proteins in one cell

Question 35

What is the difference between a monoclonal and polyclonal antibody?

Answer 35

Monoclonal = high specificity, single epitope on protein, weaker signal

polyclonal = less specifity (more BG noise) multiple epitopes, stronger signal

Question 36

What is the function of the fixative, pectolyase/cellulase, and Triton-X 100 in this procedure

Answer 36

fixative: Preserves cell structure by cross-linking proteins.

pectolyase/cellulase: Partially digests cell walls to increase antibody access.

Permeabilizes cell membranes to allow antibodies to enter cells.

Question 37

How can you fluorescently label two proteins simultaneously in one cell?

Answer 37

use primary antibodies raised in different species, and secondary antibodies tagged with distinct fluorophores.