Lecture 12 - biochem analysis pt.2 Flashcards

1
Q

what moves faster thruogh PAGE and what direction

A

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

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2
Q

struct of SDS

A

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

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3
Q

what is a detergant moleucle

A

has dual character
an ionic and hydrophobic part

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4
Q

what struc does SDS form in aq soln

A

micelles

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5
Q

what must be doen to proteins before adding SDS

A

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

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6
Q

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

A

1.4g SDS per gram of protein

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7
Q

what is size of SDS-protein complex proportional to

A

the molecular weight of the protein

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8
Q

how to use SDS PAGE to find molecular weight of protein

A

with molecular weight markers

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9
Q

why is SDS needed in the first place

A

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

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10
Q

How to measure protein purity with sds

A

diff bands separated in the lanes
One band = pure

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11
Q

How to measure abundance of the protein

A

band intensity of the stain

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12
Q

what are the AAs that can be protonated

A

Lys
Arg
His

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13
Q

what are AAs that can be deporotonated

A

Glu
Asp

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14
Q

what is isoelectric point

A

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

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15
Q

what happens at low pH to a protien

A

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

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16
Q

what happens at high pH

A

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

17
Q

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

A

when they are neutral
so they accumulate at their pI

18
Q

what is 22D gel electrophoresis

A

combining a isoelectric focusing result with SDS PAGE

19
Q

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

A

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

20
Q

what can give us accurate mass determination of a protien

A

mass spectrometry

21
Q

what is step 1 and 2 of of mass spec

A

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

22
Q

3rd step of mass spec

A

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

23
Q

step 4 and 5 of mass spec

A

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

24
Q

what is m/z

A

mass to charge ratio of the peptide

25
what phase does mass spectrometry need to be done in
gas phase
26
what can proteomics be used to show
differential expression between: - diff cell types - diff diseases - cells treated or not treated with drugs etc
27
what domain is the heavy chain
Fc domain
28
which antibody type preffered in biochem techniques
monoclonal antibodies cuz more specific
29
immunoaffinity chromatography how
immobilise monoclonal antibody protein passed through prtien of interest gets trapped
30
why is elution hard in immunoaffinit chrom
vry tight bindng so have to be drasric like denaturing the proteins or changing pH
31
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
32
what is actin filament labelled with
RFP
33
what are microtubules labelled with
GFP
34
what are nuclei labelled with
blue fluorescent dye DAPI
35
what technique is immunogold labelling used with and why
electron microscopy cuz gold particles have high e- density
36
What usually used to break up disulphide bonds
mercaptoethanol
37
Common first step in proteomic analysis
digesting protein with protease e.g. Trypsin
38
Where does trypsin cleave
After lysine and arginine
39
What salt used to separate proteins based on their solubility
Ammonium sulphate This is part of lecture 11 btw