Lecture 6 Method II

Question 1

column chromatography

Answer 1

separation of proteins using a solid matrix,
three types of column chromatography:
- based on size
- based in charge
- based on affinity
(we often use two or three for the same sample)

Question 2

tags for protein purification

Answer 2

alternative method to column chromatography, eg 6xHis

Question 3

proteins on SDS-PAGE (electrophoresis)

Answer 3

SDS gives negative charge to the proteins, separation based on size

Question 4

isoelectric focusing (IEF)

Answer 4

separation based on charge, using a PH gradient

Question 5

two-dimensional SDS-PAGE

Answer 5

combining separation based on size/shape and charge

Question 6

western blot on a 2D protein gel

Answer 6

first electrophoresis, then put on a membrane, then antibodies detecting protein of interest

Question 7

mass spectrometry

Answer 7

identify and detect proteins by precisely measure their mass

process: vaporize the sample, ionize, accelerate through electric field, magnetic field (sort according to mass-to-charge ratio (m/z)), detection

Question 8

X-ray cristallograhpy

Answer 8

determination 3D-structure of proteins

crystallization of proteins, X-ray diffraction different depending in the 3D-structure

Question 9

Nuclear Magnetic Resonance (NMR) spectroscopy

Answer 9

determination of 3D-structure of proteins

in solution, using the spin, electromagnetic field

Question 10

(Protein) sequence alignment

Answer 10

comparison of sequences of different proteins, conserved structures correspond to important motifs

Question 11

Fluorescence resonance energy transfer (FRET)

Answer 11

see if proteins interact

eg one protein returns blue light emission when excited with violet light, another emitts green light when excited by blue light
-> if the proteins interact we will have a green light emission when the sample is exposed to violet light

Question 12

DNA gel electrophoresis

Answer 12

similar to protein electrophoresis, except DNA already negatively charged

cur with restriction enzymes

Question 13

labeling of DNA

Answer 13

incorporation of nucleotides that are detectable

eg:

• radiolabel
• fluorescente label

Question 14

DNA libraries

Answer 14

DNA fragment inserted into a plasmid, introduced to a bacterial cell, cell colony produces thousands of copy

two types of libraries:

• genomic DNA (DNA fragments including introns and non transcribed DNA)
• cDNA (includes transcription of DNA, introns removed)

Question 15

DNA sequencing

Answer 15

ddNTP instead of dNTP, can connect but no more nucleotides can add on to the chain

we get different lengths of sequences that are run on a gel

manual or automated

Question 16

open reading frames (ORFs)

Answer 16

different codons (set of three nucleotides) correspond to different amino acids

-> DNA sequence can be read in different ways depending on where you start

an open reading frame (ORF) is the part of a reading frame that has the ability to be translated

An ORF is a continuous stretch of codons that may begin with a start codon (usually AUG) and ends at a stop codon (usually UAA, UAG or UGA)

Question 17

gene organization

Answer 17

promoter, regulatory sequences, DNA, gene, transcription starts, transcription ends, mRNA, ribosome binding site, translation starts, translation stops