Protein Expression Flashcards
How are proteins synthesized (translated from mRNA) in a cell?
- Transcription of DNA to mRNA
- Transport of mRNA to cytoplasm through Nuclear Pore Complex
- Ribosome quality checks mRNA
- Ribosome translates good mRNA into a chain of amino acids using tRNAs (Based on triplet codon)
- Newly formed polypeptide chain is folded into protein by chaperones
- Functional tertiary structure of protein is formed
What happens if there is an error in the translated protein?
Can form toxic compound due to improper folding.
Immediately degraded by proteosomes.
How is mRNA formed
- RNA Pol II copies DNA info into pre-mRNA
- Pre-mRNA undergoes 5’ capping and 3’ polyadenylation and splicing in the nucleus
How is Western Blot used to compare the relative protein expression between two samples?
Protein Expression is dependent on Banding density, higher density means more of the expressed protein
How is immunofluorescence used to compare differences in protein expression between two samples?
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.
How is immunofluorescence used to compare differences in protein localization between two samples?
Immunofluorescence highlights protein localization by using fluorescently labeled antibodies targeting specific proteins. Comparing the fluorescence patterns between two samples reveals differences in protein localization.
How is immunohistochemistry used to compare differences in protein expression from two tissue samples?
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.
How are fusion proteins (e.g. A protein fused to GFP) used to visualize protein expression and localization in a cell?
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.
How are protein biomarkers or chemical fluorescent tags used to mark an organelle in a cell (e.g. mitochondria, nucleus)?
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.
Compare and contrast Prokaryote and Eukaryote Gene structure
Eukaryotes: Monocistronic (one gene = one protein)
Prokaryotes: Polycistronic (one operon = several proteins)
Both have promoter regulating the gene and coding sequence (structural gene/s)
T/F Prokaryotes sometimes only need one promoter. Defend your answer
True, this is because prokaryote genes are very compact to be efficient, for simultaneous expression hence only one promoter
Discuss the transcription initiation process w/ the TATA box
Tata Binding protein binds to TATA box
Transcription factor forms complex making RNA Polymerase (Binds RNA Pol II)
Differentiate Gene Expression in Prokaryotes vs Eukaryotes?
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
In Pre-mRNA processing, produces multiple mRNA from a single gene due to variable intron removal
Alternative Splicing
Which has 70S and 80S ribosome units pro- or eukaryote? What are their respective sub units?
Pro: 70S (50S + 30S)
Eu: 80S (60S + 40S)
Unit for Ribosomal units is S or Svedberg, what does it reflect?
Density -> Density changes when structures combine which is why 50+30=70 and 60+40=80
Occurs at cysteine in DNA and Uracil in protein
Methylation
T/F Methylation has no effect in histones?
False, Histone methylation can cause heterochromatin to be active or euchromatin to be inactive
Regulation of transcription is done by codons from and to upstream or downstream? Do they inhibit or promote transcription?
Upstream regulates downstream. They can enhancers (transcription factors) or silencers (inhibitor)
Also called Western Blot Analysis, this is a method to study Protein Expression that separates proteins based on size
SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
What allows proteins to move through the gradient in Western Blot Analysis?
SDS (Sodium Dodecyl Sulfate) - Sulfate makes it negative
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?
Colorimetric
Chemiluminescence
Fluorescence
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
Colorimetric
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.
Chemiluminescence
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
Fluorescence
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?
Lack of WRN decreases Lamin B1 but not Lamin A/C or Lap2 expression relative to WT cells (has WRN)
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)
Enzyme-linked immunosorbent assay (ELISA)
- Target molecules (e.g., proteins) are immobilized on the surface of the wells.
- Unbound sites are blocked to prevent non-specific binding.
- A specific antibody is added, binding to the target molecule.
- A secondary enzyme-linked antibody is introduced, binding to the primary antibody.
- A substrate for the enzyme is added, generating a detectable signal (e.g., color change).
- The intensity of the signal is measured, correlating with the concentration of the target molecule.
This is the limitation of ELISA
In solution, meaning liquid, also limited to what the antibody will bind to.
Main methods of protein expression study in a tissue?
Co-immunofluorescence
Enzyme based (HRP-DAB)
Gold-labelling
Method to observe protein expression where the histological slide, if colored brown, shows the target protein
HRP-DAB
No stain needed, relies on reflection of heavy molecules
Gold-labelling, uses SEM
Method that uses fluorescence to show different proteins in the tissue
Co-immunofluorescence
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?
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
