L1-sds/western And Chip

Question 1

What is sds-page used for

Answer 1

Separation of proteins from mixture based on size alone - denatured previously by heat and dtt aswell as negative coating via sds

Run on page using current and travel to positive electrode based on size

Question 2

What is put into the loading buffer which keeps protein on gel, as well as dtt for disulfide breaking, sds, bromophenol blue

Answer 2

Glycerol dense

Question 3

How would you determine mw of it from sds

Answer 3

Measure protein distance travelled (dye)/ total dye front distance travelled

This is the rf

Then plot on x with y axis being logMW ladder

Question 4

What membrane does western blot use for transfer of proteins from gel to this (immobilised proteins)

Answer 4

Nitrocellulose membrane

Question 5

What is needed to allow transfer on nitrocellulose so yoh can use western blot to detect protein of interest AND whether there is more protein or less

Answer 5

Electric current

Question 6

How does western blot work

Answer 6

Nitrocellulose in contact with the page in between filter papers on transfer cassette

Run with electrical current to allow correct transfer

Then the proteins are immunoblotted using primary ab specific to interest protein

Then secondary is added with enzyme conjugated eg horseradish peroxidase

Chemilumiesxwnf when substrate added- light photons emitted are detected by an emission reader = thicker bands more protein

Question 7

What 2 types of buffer needed in western and why

Answer 7

Wash buffer with tris to remove unbound ab And stop non-specific binding by tris

Blocking buffer which is casein or milk powder which binds to empty spaces in membrane to block weird ab binding when added

Question 8

What housekeeping protein commonly used as a control for western and why

Answer 8

A-tubulin
Constitutively expressed in all conditions
So can be used to see if there is equal protein loading/ correct loading for each sample

Allows to determine whether the conc of protein can be compared between the samples or not

Question 9

What is an alternative to a-tubulin done before the sds

Answer 9

Bradford assaying
Colorimetric assay determining protein conc at the start using spectrophotometer against a standard curve eg with bsa

Can then load equal protein quantities before the experiment to prevent the idea more protein to begin with

Question 10

What is chip used for (chromatin immunoprecipitation)

Answer 10

Look for protein-dna interactions which determine protein function often (eg tf)

or study the epi genetic markers/histone code

Question 11

Can it be used to study other cellular events that include chromatin processing

Answer 11

Yes eg dna replication or repair

Question 12

Why are proteins often required to bind to dna eg at their promoters for example txn factors or corefularors

Answer 12

Needed to facilitate access of rna/dna polymerase since dna wrapped into chromatin fibres

Question 13

What is a nucleosome

Answer 13

147bp wrapped around histone octamers

Question 14

Give examples of modifications ptm which allow chromatin. Remodelling into hetero or euchromatin

Answer 14

Methylation via hkmt on tails eg h3k9 is repressive for txn

H3k9 acetylation however is activating

Question 15

How do tf allow this

Answer 15

Recruit these enzymes/complexes for chromatin remodelling

Allowing rna pol binding

Question 16

Give an example of this type of protein which then recruits histone modificators to allow txn

Answer 16

Estrofen receptor

Question 17

How could you use chip to analyse er binding

Answer 17

Can be used to determine if ER binds at a specific promoter for a specific gene
Eg in breast cells activated by e or without e

Question 18

What is the first step

Answer 18

Cross linking dna-protein interactions using formaldehyde (forms covalent cross links to freeze them)

Question 19

Why would you do this

Answer 19

Because tf are transient / not always bound

Question 20

What do you do next

Answer 20

Break open cells through series of buffers breaking down nuclear/cell membrane to extract chromatin

Question 21

What process is then used to fragment chromatin into small pieces but maintain the cross links

Answer 21

Sonication (breaks down phosphodiesterase bonds)

Question 22

What is the process of immunoprecipitation

Answer 22

Antibodies specific to protein of interest/ er are added with magnetic beads so can be pulled down, leaving unbound chromatin fragments in supernatant

Question 23

Why is it important to have a control here and what is used

Answer 23

IgG with nonspecific binding capabilities is added so you can distinguish specific ab interactions vs non specific immunoprecipitation

This is because beads may bind unrelated proteins/ some proteins could be pelleted which aren’t protein of interest causing false positives

Question 24

When would you go ahead with the data analysis in this case

Answer 24

If there is more specific binding than igG background % binding

Question 25

What are the final steps before sequence analysis

Answer 25

Cross links/complexes are washed reversed using heat and salt buffer

Then you degrade the proteins using proteinase K leaving you with just the chromatin fragment of interest

Question 26

What 2 ways could you use to analyse the data

Answer 26

Qpcr where you make primers flanking 3’ and 5’ of the sequence eg promoter

Or you can send it off for chip sequencing and analyse protein interactions across whole genome/ epi genetic markers across whole genome

Question 27

What needs to be done to dna before PCR

Answer 27

Purification which is the removal of protein and salt

Question 28

What sort of PCR wojld you expect in no presence of esteofen

Answer 28

No binding of er to the promoter and therefore no PCR product because lack of immunoprecipitation

Question 29

How could you tailor this experiment to see if er binding to promoter is associated with methylation ie txn regression or txn activation via acetylation at specific markers h3k9

Answer 29

You would do chip with ab specific for h3k9 me or ac

If there is these markers then when you do PCR of this region in the chromatin then there will be more product

Can compare in absence or preeence of er