Molecular - protein analysis

Question 1

How does proteome compare to transcriptome?

Answer 1

Splicing; regulation of translation; lifespan of mRNA vs protein; processing on mRNA

Question 2

Alternative splicing

Answer 2

regulated by cis acting elements (splicing enhancer and silencer) and transacting RNA binding proteins (hnRNPs silence, SR proteins activate)

Question 3

Splicing in transformer (tra)

Answer 3

Sxl (sex lethal - only in female) silences early exon 2 splice site in females (which contains a stop codon), so Tra is produced. In males the site is recognized by a factor (U2 associated factor) and results in truncated protein

Question 4

Splicing in dsx (determination of sex)

Answer 4

TF for sex differentiation. Tra acts as nucleation site with Tra2 on exon 4 so that it gets transcribed. Only happens in females, so exons expressed are 1,2,3,4 which activates female genes. Exon 4 has polyA tail, other exons not added to it. In males it skips exon 4 and we get 1,2,3,5,6.

Question 5

Typical processing

Answer 5

Spliceosome removes introns; intron forms a loop called a lariat structure

Question 6

Spliceosome contains

Answer 6

U1, 2, 3. Associated factors.

Question 7

Protein modification

Answer 7

Phosphorylation (kinase, with phosphatase opposite); glycosylation/tagging/recognition; cleavage (activating zymogens); methylation; Metals/cofactors; acetylation (histones condense); ubiquitination

Question 8

Glycosylation

Answer 8

Aids in folding, cell-cell adhesion.

Question 9

Acetylation

Answer 9

Turn on or off.

Question 10

Phosphorylation

Answer 10

Kinase adds a (P), phosphorylase removes. (P) generally activates

Question 11

Ubiquitination

Answer 11

signal for their degradation via the proteasome, alter their cellular location, affect their activity and promote or prevent protein interactions

Question 12

Chromatography origin

Answer 12

“Colour writing”; made by Tsvet in 1903 with plant pigments interacting with chalk and alumina

Question 13

HPLC

Answer 13

High performance liquid chromatography. Pump pushes solvent through column (high pressure), detector displays at computer. (could also filter a little off for mass spec).

Question 14

HPLC variation

Answer 14

Two solvent system. Start with solvent 1, then gradually add (mix) solvent 2. For example, nonpolar substance in water binds to column, then gradually reduce water and increase nonpolar solvent, gradual separation.

Question 15

Normal phase

Answer 15

Stationary phase is polar, solvent is nonpolar.

Question 16

Reverse phase

Answer 16

Solid phase is nonpolar, solvent is polar. More common

Question 17

Solid phase examples

Answer 17

The packing material. Polar to nonpolar: Silica, CN, C8, C18 (C’s added onto the O’s in silica).

Question 18

Solvent choices

Answer 18

Polar to nonpolar: salts, acids, bases, alcohols, hydrocarbons (hexane). Trial and error, depends what you’re purifying.

Question 19

Separation

Answer 19

Determined by length of column (more material and solvent - more money but better separation) and bead size (smaller (5-10µm) gives more precise but needs increased pressure)

Question 20

Ion exchange

Answer 20

Anionic or cationic groups immobilized on matrix (depend on the beads). More highly charged are bound tightest, so slowest.

Question 21

Differing conditions - how would characteristics of solute influence choice of solid phase and buffer pH?

Answer 21

See table. For isolating weak acids/bases we manipulate them, for strong acid/base we manipulate the matrix

Question 22

Anion exchanger

Answer 22

It is a cation. Tricksy.

Question 23

Gel Filtration

Answer 23

Gel with beads with specific pore size. Small molecules can enter, huge path length, therefore the larger ones move faster

Question 24

Use of gel filtration

Answer 24

Purify PCR product. Primer and nucleotides will be slowed/trapped in the beads, DNA strands will pass quickly. Ex. Sephadex G50 - excludes anything >50bp

Question 25

Affinity chromatography

Answer 25

Antibodies bound to the beads, antigens binds to it while others pass through, then add low pH to release the antigen. The opposite works too to separate antibodies. Very precise.