L22 - Functional genomics 2 Flashcards
(19 cards)
define isolectric point
The pH at which the protein becomes uncharged
= will no longer move in elctrophoresis gel
what 2 physical properties dowe use to seperate proteins to analyse THE PROTEOME OF A CELL
- Isoloelectric point
- molecular mass
what does the isoelctric point depend on
sequence of amino acids in the protein
describe 2D gel electrophoresis method
- mix of proteins in acidic buffer added to tube of gel
= acidic buffer means most proteins are positively charged
- pH gradient is inside tube with high pH/low H+ ions at bottom
- negative charge at bottom of tube applied by electric current
= proteins migrate topH that represents there isoelectric point (pI)
what does 2D gel elctrophoresis seperate by
Isolectric point - pI
= not by size or charge
= chnge in abundance of H+/protons further down the tube due to higher pH cause proteuns to be less positively charged as they move down the gel
why do proteins stop in 2D gel elctrophoresis
isoelectric point reached
= no longer charged due to lower number of available H+ to make positive
what is added for the next step after 2D gel elctrophoresis
SDS
= negatively charged detergent
= the now negativel;y charged proteins are put onto SDS-acrylamide gel and seperated by size
what does the SDS in the second stage of protein seperation produce
3d map = proteins ahvce been seperated by 2 dimensions
- molecular wight and pH
how are the proteins visulaised in 2D and SDS analysis
silver staining
= very small quantities of protein
why is a lot of clustering seen when looking at 3D map of proteins
lots of proteins have similar isolectric points and molecular masses
= cluster around pH 7 for example
= makes it difficult to quantify how mcuh of each protein
why do membrane proteins not work for 2D and 3D protein analysis
need soluble proteins
why does 2d cell electrophorsis have low dynamic range
limited by amount od dye present
= high protein level is not all stained
what is DIGE
running 2 samples - either of digffrent people or different conditions - on the same gel
the 2 samples are flourescemtly tagged with different colours
= the different range of colours allow us to compare the samples for protein expression
= is one sample producing more of a certain protein than another ?
what do Cy3 and Cy5 tag in terms of colour
floursecnt tags
Cy3 = green
Cy5 = red
what do we use to flourescently tag the proteins in DIGE
tag to specifc amino acids
= we’re not trying to identify specific sequyences we just want all the proteins in the sample to be tagged
= Cy3 or Cy5 used
why can we not compare the intensity of flourescence in proteins in DIGE
not all proteins have the same amount of lysines
= less or more tags bound affect the strength of flourescence
= we can however compare BETWEEN samples as the tagging for a protein would be the same regardless of individual
descibe the method of spot identification
- protein spot is picked by robot and digested by protease - Trypsin
= akways cuts at same points - lysines and aragnines
- cut sample is passed ionto mass spectrometer and mass of the created fragments measured
= compare Mr of fragments from Mass spec to known database
= is there more or less of the ‘expected’ proteins
describe Liquid chromatography-Mass spec/Mass-spec method
- proteins seperated by SDS-PAGE
- proteins digestisted by protease/trypsin
= peptide fragments produced
- peptide fragments seperated by liquid chromatography
= meausres abundance of each peptide fragment
= detector at bottom of collumn as they come out = produces peaks
- double mass-spec (MS/MS)
- gives the Mr/mass of the proteins
- breaks the peptides up into even smaller fragments by physical force = different smaller sized fragments
= sequence of proteins can be worked out based on the breaks caused and comparing to known peptides in database
why is LC-MS/MS an advcanced version of identifying proteins to 2D electrophoresis
proteins are digested before you seperate them
= wider range of proteins due to highly positiove or nefgative and membraneproteins being included
alternative cell types or samples can be run through this method and then results compared to see difference in gene expression/proteins present
