Molecular Seperations Flashcards Preview

Biochem - Year 1 Biomed > Molecular Seperations > Flashcards

Flashcards in Molecular Seperations Deck (35)
Loading flashcards...
1
Q

Why do we purify molecules

A

Study properties
Analyse the distribution in body
Use them eg for medical purpose

2
Q

What are the 2 techniques used to seperate molecules

A

Chromatography

Gel electrophoresis

3
Q

How do you acctually prepare intracellular proteins before using chromatography

A

Using a centrifuge you isolate proteins from the rest of the cell debris eg membrane

Spin at high speeds and the pellet will be cell debris, the supernatant is the proteins/molecules you want to seperate fully

4
Q

What is g force in terms of centrifugation / ultracentrifugation

A

Acceleration due to gravity (speed = centrifugal force)

5
Q

What are 4 properties chromatography exploits to seperate proteins

A

Charges

Size/shape

Affinity (binding site)

Hydrophobicity

6
Q

What is the mobile and stationary phase meaning

A

Mobile phase is the phase in which molecules move (water usually)

Stationary phase is the phase in which molecules don’t move (eg column or paper)

When molecules are in chromatography they pass the stationary phase and those that bind don’t stay mobile phase. Proteins/molecules that keep moving down are in the mobile phase

7
Q

What are the 2 forms of chromatography

A

Thin layer chromatography- using paper

Column chromatography- much longer mobile phase - seperate at a bigger scale.

8
Q

How do molecules move up in TL chromatography

A

Molecules soluble in the Eg water are part of solvent and move up with it as a solvent

9
Q

What are the molecules/proteins called when they come out of the column (don’t bind to stationary phase)

A

The eluate

The protein is eluting down the column

10
Q

How does size exclusion chromatography seperate proteins (gel filtration)

A

Porous bead are added to the column
Small molecules move into the beads but larger molecules pass by

Larger molecules = move faster (elute)

11
Q

What is chromatography which used charges on proteins to seperate called and how does it work

A

Ion exchange chromatography

If the stationary phase is +ve , proteins with a negative net charge (side chains) will bind to stationary phase

-ve proteins therefore elute

12
Q

How do you overcome ion exchange chromatography to get the protein you want

A

Add salt (naCl-) which means cl will compete to bind to stationary phase ,

The proteins then elute

13
Q

Explain the reverse phase chromatography process (using hydrophobicity)

A

Proteins with hydrophobic side chains will bind the the surface due to water (polar). This means proteins that aren’t hydrophobic will elute

14
Q

How do you overcome hydrophobic proteins sticking to the stationary phase

A

Use ethanol non polar solvent instead

15
Q

How does affinity binding chromatography work

A

Molecules with specific binding sites such as antibodies will bind to the stationary phase if there are beads attached to the complementary binding site

They therefore stay in the column

16
Q

How do you overcome affinity binding chromatography

A

Add free binding proteins/molecules to bind to the molecules instead of the beads , this allows them to elute

17
Q

What does IMac stand for and what is it used for

A

Immobilised metal affinity chromatography.

It is used for proteins that are harder to seperate

18
Q

How are recombinant proteins used in IMAC to seperate proteins

A

The proteins are engineered to have added 6-8 histidine residues(bind with metal)

This allows binding to metal such as zinc++ on the column

19
Q

How is iMac proteins eluted when they’ve bonded with metals

A

Add imidazole (structure like histidine) which allows histidine to detach and elute the protein

20
Q

How is gel electrophoresis different to chromatography

A

No mobile phase- the liquid is in gel
A gel is used to prevent convection currents (no water circling)

The gel allows movement as bands

21
Q

What controls the speed of molecules in the gel

A

Pore size of gels. There are usually different sized to stop small molecules moving too fast

22
Q

What is induced to allow movement in gel electrophoresis

A

Electric fields. There will be either or both positive or negative electrodes

23
Q

What is PAGE

A

Polyacrylimide gel electrophoresis

24
Q

What does NATIVE PAGE mean

A

Proteins seperated by size shape and charge

25
Q

Why are proteins all usually kept at ph 8

A

so that all proteins have a -ve net charge (lose protons)

This allows for the movement in one direction

Also any higher would denature the proteins

26
Q

Explain what SDS page is and how it works

A

SDS molecule is added which adds a negative overall charge of all proteins

It means all proteins are seperated by size not charge

27
Q

What is added in sds page to break disulfide bonds in proteins

A

Mercapto ethanol and they’re heated

28
Q

Does smaller proteins go further in electrophoresis (sds)

A

Yes, larger proteins appear first

29
Q

How do you calculate the molecular weight from SDS page

A

Use log 10 because it’s in a log scale and then x by the scale the protein shows on = MW

30
Q

What is the gel electrophoresis called when proteins are seperated by charges at certain ph

A

Iso electric focussing

31
Q

In IEF what is the point where the protein appears in the gel

A

The isoelectric point

Where proteins negative and positive charges are equal at a certain ph

No net charge

32
Q

What is the ph gradient produced by in ief

A

Ampholytes

33
Q

2D gel electrophoresis uses both ief and SDS. Explain the process

A

First the proteins are seperated by ief using the ph gradient
Once Ip is found
They are put in an electric field with a positive electrode at the bottom

The proteins then seperate using size (smallest goes the furthest)

34
Q

Why would more proteins appear in SDS than ief

A

Some proteins have the same isoelectric point

35
Q

Why is adding ethanol to stationary phase called reverse phase hydrophobicity chromatography

A

Because it is polar so elutes the hydrophobic proteins eventually