Protein Expression

Question 1

How are proteins synthesized (translated from mRNA) in a cell?

Answer 1

1. Transcription of DNA to mRNA
2. Transport of mRNA to cytoplasm through Nuclear Pore Complex
3. Ribosome quality checks mRNA
4. Ribosome translates good mRNA into a chain of amino acids using tRNAs (Based on triplet codon)
5. Newly formed polypeptide chain is folded into protein by chaperones
6. Functional tertiary structure of protein is formed

Question 2

What happens if there is an error in the translated protein?

Answer 2

Can form toxic compound due to improper folding.
Immediately degraded by proteosomes.

Question 3

How is mRNA formed

Answer 3

1. RNA Pol II copies DNA info into pre-mRNA
2. Pre-mRNA undergoes 5’ capping and 3’ polyadenylation and splicing in the nucleus

Question 4

How is Western Blot used to compare the relative protein expression between two samples?

Answer 4

Protein Expression is dependent on Banding density, higher density means more of the expressed protein

Question 5

How is immunofluorescence used to compare differences in protein expression between two samples?

Answer 5

Immunofluorescence uses fluorescently labeled antibodies to bind to specific proteins in samples. By comparing the intensity or distribution of fluorescence between two samples, differences in protein expression can be inferred.

Question 6

How is immunofluorescence used to compare differences in protein localization between two samples?

Answer 6

Immunofluorescence highlights protein localization by using fluorescently labeled antibodies targeting specific proteins. Comparing the fluorescence patterns between two samples reveals differences in protein localization.

Question 7

How is immunohistochemistry used to compare differences in protein expression from two tissue samples?

Answer 7

Immunohistochemistry (IHC) utilizes antibodies to detect specific proteins in tissue samples. By comparing the staining intensity or distribution of proteins between two tissue samples, differences in protein expression can be assessed.

Question 8

How are fusion proteins (e.g. A protein fused to GFP) used to visualize protein expression and localization in a cell?

Answer 8

Fusion proteins, like a protein fused to green fluorescent protein (GFP), are used to visualize protein expression and localization in cells. GFP emits green fluorescence when exposed to certain light wavelengths. By fusing GFP to a protein of interest, researchers can track the location and abundance of the protein within cells using fluorescence microscopy.

Question 9

How are protein biomarkers or chemical fluorescent tags used to mark an organelle in a cell (e.g. mitochondria, nucleus)?

Answer 9

Protein biomarkers or chemical fluorescent tags are used to mark organelles in cells by selectively binding to specific organelle structures. For example, MitoTracker binds to mitochondria, while DAPI binds to DNA in the nucleus. By labeling organelles with these markers or tags, researchers can visualize and track their localization within cells using fluorescence microscopy.

Question 10

Compare and contrast Prokaryote and Eukaryote Gene structure

Answer 10

Eukaryotes: Monocistronic (one gene = one protein)
Prokaryotes: Polycistronic (one operon = several proteins)

Both have promoter regulating the gene and coding sequence (structural gene/s)

Question 11

T/F Prokaryotes sometimes only need one promoter. Defend your answer

Answer 11

True, this is because prokaryote genes are very compact to be efficient, for simultaneous expression hence only one promoter

Question 12

Discuss the transcription initiation process w/ the TATA box

Answer 12

Tata Binding protein binds to TATA box
Transcription factor forms complex making RNA Polymerase (Binds RNA Pol II)

Question 13

Differentiate Gene Expression in Prokaryotes vs Eukaryotes?

Answer 13

Pro: Ribosomes are readily available to transcribe mRNA since no nuclear envelope

Eu: pre-mRNA needs to be processed first then transported out of the nucleus as mRNA to be processed by the Ribosomes

Question 14

In Pre-mRNA processing, produces multiple mRNA from a single gene due to variable intron removal

Answer 14

Alternative Splicing

Question 15

Which has 70S and 80S ribosome units pro- or eukaryote? What are their respective sub units?

Answer 15

Pro: 70S (50S + 30S)
Eu: 80S (60S + 40S)

Question 16

Unit for Ribosomal units is S or Svedberg, what does it reflect?

Answer 16

Density -> Density changes when structures combine which is why 50+30=70 and 60+40=80

Question 17

Occurs at cysteine in DNA and Uracil in protein

Answer 17

Methylation

Question 18

T/F Methylation has no effect in histones?

Answer 18

False, Histone methylation can cause heterochromatin to be active or euchromatin to be inactive

Question 19

Regulation of transcription is done by codons from and to upstream or downstream? Do they inhibit or promote transcription?

Answer 19

Upstream regulates downstream. They can enhancers (transcription factors) or silencers (inhibitor)

Question 20

Also called Western Blot Analysis, this is a method to study Protein Expression that separates proteins based on size

Answer 20

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)

Question 21

What allows proteins to move through the gradient in Western Blot Analysis?

Answer 21

SDS (Sodium Dodecyl Sulfate) - Sulfate makes it negative

Question 22

After SDS-PAGE, electrophoretic protein transport is done to transfer it into a blotting membrane for staining. In western blot there are three methods of staining detection, what are they?

Answer 22

Colorimetric
Chemiluminescence
Fluorescence

Question 23

Western blot relies on antibody staining this method of detection makes use of a substrate that binds to the antibody producing a product without light. Usually permanent

Answer 23

Colorimetric

Question 24

Western blot relies on antibody staining this method of detection makes use of a substrate that binds to the antibody producing a product and light. Gives of light.

Answer 24

Chemiluminescence

Question 25

Western blot relies on antibody staining this method of detection makes use of a substrate that binds to the antibody producing light only. No need for substrate and needs a machine like a scanner for excitation and emmision

Answer 25

Fluorescence

Question 26

See sample exercise in the first PPT for the figure:

Western blot analysis of lamin B1 from extracts of cells with or without WRN gene (gene knockout of WRN). Antibody reactions against normalized cell extract are shown for lamin B1, laminA/C and the lamin-binding protein Lap2β.

In the blot where WRN was removed, Bands for all except Lamin B1 stayed the same. Lamin B1 faded. What does this mean?

Answer 26

Lack of WRN decreases Lamin B1 but not Lamin A/C or Lap2 expression relative to WT cells (has WRN)

Question 27

Used to quantify proteins in a solution using antibodies that captures the protein target forming a product you can use in assay (absorbance measurement in 96 well plate)

Answer 27

Enzyme-linked immunosorbent assay (ELISA)

1. Target molecules (e.g., proteins) are immobilized on the surface of the wells.
2. Unbound sites are blocked to prevent non-specific binding.
3. A specific antibody is added, binding to the target molecule.
4. A secondary enzyme-linked antibody is introduced, binding to the primary antibody.
5. A substrate for the enzyme is added, generating a detectable signal (e.g., color change).
6. The intensity of the signal is measured, correlating with the concentration of the target molecule.

Question 28

This is the limitation of ELISA

Answer 28

In solution, meaning liquid, also limited to what the antibody will bind to.

Question 29

Main methods of protein expression study in a tissue?

Answer 29

Co-immunofluorescence
Enzyme based (HRP-DAB)
Gold-labelling

Question 30

Method to observe protein expression where the histological slide, if colored brown, shows the target protein

Answer 30

HRP-DAB

Question 31

No stain needed, relies on reflection of heavy molecules

Answer 31

Gold-labelling, uses SEM

Question 32

Method that uses fluorescence to show different proteins in the tissue

Answer 32

Co-immunofluorescence

Question 33

Look at the figure in Protein Expression Cont. pdf:

Comparing the SGLT2 using HRP-DAB in normal and diabetic nephropathy kidney biopsies, you can see that there are darker brown stains in the epithelial kidney cells of the diabetic sample’s histo slide, what does this imply?

Answer 33

Since it is darker, that means it has more of the protein tagged by the stain. This therefore means that SGLT2 is overexpressed in kidney epithelial cells of diabetic patients