03 Flashcards
Creation of a Functional Protein steps
- Synthesis
- Protein Folding (and cofactor binding, noncovalent interactions)
- Covalent Modification (phosphorylation, acetylation, etc)
- Binding of other cofactors or proteins
Covalent modification of proteins occurs by ______ + is necessary to elicit a ______ _________ response
enzymes, specific biological
Ubiquitin
A very large protein made up of 76 amino acids
When put on proteins, its COOH attaches to lysine amino acids side chains
Ubiquitination
marking proteins for degradation
Ubiquitination Process I
E1 enzyme picks up ubiquitin
E2/E3 complex transfers ubiquitin to target protein
Protein is marked for degradation
SUMO
A small protein molecule, ~100 amino acids
4 SUMO molecules found in mammals
PCR Steps
Denaturation (95 degrees) → Hydrogen bonds break, DNA strands separate
Annealing (50-60 degrees) → Primers hydrogen bind to the DNA
Extension (72 degrees) → DNA polymerase extends/synthesizes/copies the DNA
What is CT?
(Cycle threshold) = cycle # where fluorescence crosses a set threshold
Lower CT → ____ starting DNA
More
Higher CT → ____ starting DNA
Less
RT-rt-PCR (Real Time reverse transcriptase PCR )
Performs reverse transcription and PCR reactions separately to determine the quantity of RNA in a sample
Reverse transcriptase takes RNA and makes cDNA copy → now can do PCR with it
Cannot do PCR or RT-rt-PCR with RNA
Converts RNA → cDNA, then amplifies it.
Measures RNA levels in a sample.
Cycle Threshold (CT)
Based on the detection and quantitation of a fluorescent reporter
The first significant increase in the amount of PCR product (CT - threshold cycle) correlates to the initial amount of target template
Sumoylation
Similar to ubiquitination but serves different regulatory roles.
Can be example of duplication/divergence
Results in different functional changes in proteins
Conjugation (way it attaches to proteins) is similar to Ubiquitin/ation
Steps to Sumoylation
SUMO is activated by E1 enzyme
Activated SUMO is transferred to E2 enzyme
E3 enzyme (SUMO ligase) facilitates the attachment of SUMO to a specific lysine residue on the target protein
PCR
Take DNA, add components (nucleotides, triphosphates, primers) to amplify DNA
Point: to amplify DNA for a purpose (i.e. cloning, analysis, etc)
Go through 3 steps and repeat/cycle → increase DNA by a million fold
What is wrong with Agarose Gels?
Poor precision
Low sensitivity; can’t determine how much DNA was there to start with
Short dynamic range < 2 logs
Low resolution
Non-automated → time consuming
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide staining is not very quantitative
