Lecture 12 - biochem analysis pt.2

Question 1

what moves faster thruogh PAGE and what direction

Answer 1

-ve proteins move towards +ve electrode
smaller ones move faster through gel

Question 2

struct of SDS

Answer 2

sodium dodecyl sulphate
basically a fatty acid with a sulphate group
CH3-(CH2)11-SO4^-Na+

Question 3

what is a detergant moleucle

Answer 3

has dual character
an ionic and hydrophobic part

Question 4

what struc does SDS form in aq soln

Answer 4

micelles

Question 5

what must be doen to proteins before adding SDS

Answer 5

linearise it
and denature it
since SDS only forms complex with denatured proteins

Question 6

what is the ratio of SDS/g of protein when theyre combined

Answer 6

1.4g SDS per gram of protein

Question 7

what is size of SDS-protein complex proportional to

Answer 7

the molecular weight of the protein

Question 8

how to use SDS PAGE to find molecular weight of protein

Answer 8

with molecular weight markers

Question 9

why is SDS needed in the first place

Answer 9

so that the proteins charge becomes irrelevant and its only separe]ated based on its size

Question 10

How to measure protein purity with sds

Answer 10

diff bands separated in the lanes
One band = pure

Question 11

How to measure abundance of the protein

Answer 11

band intensity of the stain

Question 12

what are the AAs that can be protonated

Answer 12

Lys
Arg
His

Question 13

what are AAs that can be deporotonated

Answer 13

Glu
Asp

Question 14

what is isoelectric point

Answer 14

the pH at which the protein is neutrally charged
(pI)

Question 15

what happens at low pH to a protien

Answer 15

Glu and Asp will keep their proton
Lys Arg and His get protonated
so overall positive charge

Question 16

what happens at high pH

Answer 16

the opposite
except Lys Arg and His stay neutral
and Glu and Asp are -ve
so overall -ve protein

Question 17

in isoelectric focusing, at what point do the protiens become stationary on the medium

Answer 17

when they are neutral
so they accumulate at their pI

Question 18

what is 22D gel electrophoresis

Answer 18

combining a isoelectric focusing result with SDS PAGE

Question 19

what does x and y axis of a 2d electrophoresis gel represent

Answer 19

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

Question 20

what can give us accurate mass determination of a protien

Answer 20

mass spectrometry

Question 21

what is step 1 and 2 of of mass spec

Answer 21

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

Question 22

3rd step of mass spec

Answer 22

peptide separation
using hydrophobicity chromatography, the protein is spearated into peptides
each stick = 1 peptide

Question 23

step 4 and 5 of mass spec

Answer 23

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

Question 24

what is m/z

Answer 24

mass to charge ratio of the peptide

Question 25

what phase does mass spectrometry need to be done in

Answer 25

gas phase

Question 26

what can proteomics be used to show

Answer 26

differential expression between:
- diff cell types
- diff diseases
- cells treated or not treated with drugs
etc

Question 27

what domain is the heavy chain

Answer 27

Fc domain

Question 28

which antibody type preffered in biochem techniques

Answer 28

monoclonal antibodies
cuz more specific

Question 29

immunoaffinity chromatography how

Answer 29

immobilise monoclonal antibody
protein passed through
prtien of interest gets trapped

Question 30

why is elution hard in immunoaffinit chrom

Answer 30

vry tight bindng
so have to be drasric
like denaturing the proteins or changing pH

Question 31

western blot analysis steps

Answer 31

• electrophoresis to separate proteins
• transfer proteins onto medium
• use radioactive/enzyme/luminescent antibodies
• expose medium to antibodies
• detect the protein of interest

Question 32

what is actin filament labelled with

Answer 32

RFP

Question 33

what are microtubules labelled with

Answer 33

GFP

Question 34

what are nuclei labelled with

Answer 34

blue fluorescent dye
DAPI

Question 35

what technique is immunogold labelling used with and why

Answer 35

electron microscopy
cuz gold particles have high e- density

Question 36

What usually used to break up disulphide bonds

Answer 36

mercaptoethanol

Question 37

Common first step in proteomic analysis

Answer 37

digesting protein with protease e.g. Trypsin

Question 38

Where does trypsin cleave

Answer 38

After lysine and arginine

Question 39

What salt used to separate proteins based on their solubility

Answer 39

Ammonium sulphate
This is part of lecture 11 btw