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12 Cards in this Set

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  • Back
How can protein expression differ from mRNA expression?
Fun diff w/post translational modifications
Differential translation rates (diff. levels mRNA stability, diff. translation rates)
Differential degradation rats (can have same level of mRNA under two circumstances and one might be degraded before other does, etc)
Function dependent on protein interactions
If you had round cells and pointy cells, design an experiment to identify their proteins using 2-D GE. Any problems?
Solubilize proteins, 2-D SDS PAGE, silver stain, spots of interest excised and put in MS

Dynamic range: lowest expressed VS highest expressed protein can vary by large factors, only 10% visualized in 2-D gel
Describe protein mass mapping with mass spec. Advantage?
Trypsin digest, MALDI-TOF, database search, high throughput, automated
Describe peptide sequencing with mass spec. Advantage?
Digest, quadrapole MS (selects a peak--a peptide of certain mass--to hit wall of N2 and fragment along peptide bonds). Determine different fragments and get peptide sequences.
Not automated, not high throughput, get more info, more likely to ID
Design an experiment to identify PDGF in a protein mix.
PDGF binds PDGFR (try rec kinase)
Run gels with antibody that bonds to phosphorylated kinases under two conditions:

Compare bands and excise region that is phosphorylted only when PDGF present

Describe sequencing of individual peptides with mass spec. Advantage?
Trypsin digest, Use nanoelectrospray mass spec to get masses of individual peptides, can further fragment peptide peak via tandem MS
Design an experiment to enrich phosphopeptides can be enriched.
Trypsin digest, allows phosphopeptides to be enriched by purifying peptide mixture over metal resin microcolumn. MALDI followed by a nanoelectrospray before and after treatment with phosphatase. Will see changes in peaks (jumps) because of loss of phosphate group and can determine mass.
Describe how capillary electrophoresis works. How does it compare to 2-D gels?
Uses electric current to push proteins thorugh capillary and separate them by charge, fededing into an MS. Allows us to look at all of the occuring masses and dwell points, allowing for high resolution mass:charge ratio. Higher throughput than 2-D gels (don't need to waste time staining).
Are to MS results comparable? If not, how can these results be compared? Why would you want to do this? What is concept this known as?
No, MS signal is proportional to # of ions of given mass and ionization is extremely sensitive to conditions.

Grow cells with condition 1 in 14N source and others in 15N source, combine their proteins into a sample and run in MS (same conditions).

If you wanted to test protein ?) with cells in different conditions.

Differential display.
Design an experiment to isolate S14 interacting proteins.
S14 protein is bound to cleavable affinity tag (GST) on agarose beads in column. Protein sample incubated with beads and beads washed. Thrombin used to cleave GST-S14 connection, resulting in elution of all proteins specifically bound to S14.
Can then use 1 or 2-D GE and compare to GST alone. Excise band corresponding to target proteins and run MS.
Describe how the yeast-2 hybrid can be used to determine protein interactions.
Fuse GAL-4 BD (binds DNA) to protein to be studied ("bait")

Fuse GAL-4 AD (recruits txn factors) to cDNA library.

If no interaction, two will never associated to increase transcription f reporter gene. If prey and bait interact, increase txn of reporter gene.

Can ID the prey that got caught by sequencing the cDNA that gave us the response.
Describe the concept of phage display and how a protein microarray is related to this.
Have many many phage coat proteins fused with different peptide sequences (randomized), expose to radioactive protein of interest and see if there's interaction.

Identify clone (randomized peptide on phage) and then run through database.

protein microarray: instead of fusing random sequences you can fuse known cDNA libraries.