Lecture 12 - biochem analysis pt.2 Flashcards
what moves faster thruogh PAGE and what direction
-ve proteins move towards +ve electrode
smaller ones move faster through gel
struct of SDS
sodium dodecyl sulphate
basically a fatty acid with a sulphate group
CH3-(CH2)11-SO4^-Na+
what is a detergant moleucle
has dual character
an ionic and hydrophobic part
what struc does SDS form in aq soln
micelles
what must be doen to proteins before adding SDS
linearise it
and denature it
since SDS only forms complex with denatured proteins
what is the ratio of SDS/g of protein when theyre combined
1.4g SDS per gram of protein
what is size of SDS-protein complex proportional to
the molecular weight of the protein
how to use SDS PAGE to find molecular weight of protein
with molecular weight markers
why is SDS needed in the first place
so that the proteins charge becomes irrelevant and its only separe]ated based on its size
How to measure protein purity with sds
diff bands separated in the lanes
One band = pure
How to measure abundance of the protein
band intensity of the stain
what are the AAs that can be protonated
Lys
Arg
His
what are AAs that can be deporotonated
Glu
Asp
what is isoelectric point
the pH at which the protein is neutrally charged
(pI)
what happens at low pH to a protien
Glu and Asp will keep their proton
Lys Arg and His get protonated
so overall positive charge
what happens at high pH
the opposite
except Lys Arg and His stay neutral
and Glu and Asp are -ve
so overall -ve protein
in isoelectric focusing, at what point do the protiens become stationary on the medium
when they are neutral
so they accumulate at their pI
what is 22D gel electrophoresis
combining a isoelectric focusing result with SDS PAGE
what does x and y axis of a 2d electrophoresis gel represent
x axis will be the pI (ie the results from isoelectric focusing)
goes from low to high pI
the y axis shwos the molecular weight like a normal SDS PAGE result
what can give us accurate mass determination of a protien
mass spectrometry
what is step 1 and 2 of of mass spec
remove the band of protein ur itnerested in from the gel
then cut it using a specific protease that will cleave at specific, known points
3rd step of mass spec
peptide separation
using hydrophobicity chromatography, the protein is spearated into peptides
each stick = 1 peptide
step 4 and 5 of mass spec
each peak from peptide separation, so each peptide, is investigates by mass spec
- ions are separated based on their mass by a magnetic field
and displyed as relative abundace vs m/z
and then compared to database or computer analysed to identify protien
what is m/z
mass to charge ratio of the peptide
what phase does mass spectrometry need to be done in
gas phase
what can proteomics be used to show
differential expression between:
- diff cell types
- diff diseases
- cells treated or not treated with drugs
etc
what domain is the heavy chain
Fc domain
which antibody type preffered in biochem techniques
monoclonal antibodies
cuz more specific
immunoaffinity chromatography how
immobilise monoclonal antibody
protein passed through
prtien of interest gets trapped
why is elution hard in immunoaffinit chrom
vry tight bindng
so have to be drasric
like denaturing the proteins or changing pH
western blot analysis steps
- electrophoresis to separate proteins
- transfer proteins onto medium
- use radioactive/enzyme/luminescent antibodies
- expose medium to antibodies
- detect the protein of interest
what is actin filament labelled with
RFP
what are microtubules labelled with
GFP
what are nuclei labelled with
blue fluorescent dye
DAPI
what technique is immunogold labelling used with and why
electron microscopy
cuz gold particles have high e- density
What usually used to break up disulphide bonds
mercaptoethanol
Common first step in proteomic analysis
digesting protein with protease e.g. Trypsin
Where does trypsin cleave
After lysine and arginine
What salt used to separate proteins based on their solubility
Ammonium sulphate
This is part of lecture 11 btw
