Lecture 1-4 Flashcards
(18 cards)
How are amino acid subunit formed?
by a condensation reaction between an amino group and a carboxylic acid
What are hydrogen bond donors and acceptors?
Donors: O-H and N-H
Acceptors: N or O atom
Describe thin layer chromatography.
- Polar silica gel (stationary phase) is spread as a thin layer on a plastic sheet or glass sheet
- Samples are applied to the silica gel in a small drop of solvent, different sample spots are arranged near the button edge of the sheet
- The lower edge is dipped in a non-polar solvent and as it moves up the sheet the sample spots move
- Mobile phase is nonpolar, so nonpolar AA will move up sheet and polar will not move very much
- Sample spots are identified based on their location on the sheet
What is relative mobility?
use in thin layer chromatography, the ratio of the distance moved by the sample over the distance of the solvent from
What is column chromatogrpahy?
A column packed with silica gel that contains a sample. The a buffer flows through the column and elutes samples, samples that strongly bind to the solvent will be eluted later
What is ninhydrin?
Reacts with amino N to give purple colour
What is fluorescamine?
reacts with amino N to give yellow fluorescence
What is reversed-phase chromatography?
thin layer chromatography but uses a non-polar stationary phase and a polar mobile phase
What is ion-exchange chromatography?
Uses resins that bind to solutes according to charge:
cation exchange - resins are negative and bind to positive ions
anion exchange - resins are positive and bind to negative ions
then like charge to resin will elute first
What is metal affinity chromatography?
Column contains Ni bound to a chelating agent
His proteins will bind to Ni
If protein does not contain His, it is His tagged with 6-8 His
Imidazole buffer is added and binds to Ni which allows His to be eluted last
What is gel filtration chromatography?
Separation based on size
Stationary phase is a gel that contains beads
Small molecules will enter in and out of beads and progress through column slower
Larger molecules are excluded first
What is electrophoresis?
the separation of charged particles in an electric field
What is SDS-PolyAcrylamide Gel Electrophoresis?
A form of electrophoresis in which protein is treated with SAS so that all proteins have the same rod-like shape and negative charge, so separation is based on size
What is the isoelectric point?
the pH at which the net charge of a protein is zero
What is isoelectric focusing?
separation of proteins based on their isoelectric point using a column with a gradual decrease in pH and protein will stop when it reaches its isoelectric point
What is two-dimensional electrophoresis?
Separation of protein first by isoelectric focussing and separated based on charge and then laid on top of a SDS page gel to separate based on size
An atom with a lone pair of electrons acts an H-bond acceptor if it attracts an…
O-H or N-H
An atom with a lone pair of electrons acts as a —– if it captures an H.
base
