protein purification

Why do biomolecules need purifying

To study properties and strcutre, analyse their distribution and abundance, use the commercially or medically

What do you need to do to proteins to study them and why

Purify them to ensure results of analysis are coming from The actual protein

Why does E. coli recombinants have to be checked

The outer membrane can cause anaphylaxis

Pros and cons about native proteins

good for studying protein from its natural host, not always that much protein per cell So needs lots of cells

Pros and cons of heterozygous expression

Uses bacteria/yeast/mammalian/insect cells T o make protein fo interest, get lots of it but not always in a true native state

Why is E. coli usuefl in heterozygous expression

Can make it have 70% of its total weight as protien of interest

Why are yeast cels better than E. coli cells

They have more similar folding as eukaryotic cells

Why were whale proteins used to first study proteins

They were abundant in nature- their muscle is packed with myoglobin - meant that they could study native protiens

Biophysical properties of proteins

Size, shape, mass,strucutre,fold, interaction, charge

Isoelectric point

The pH where there is no overall charge on the protein

When are electromagnetic properties used to separate proteins

Used to measure how much protien there is, proteins ma bye fluorescent if they bind to cofactors,can bind to magnetic forms of iron mineral s

What are the two main methods of separating proteins

Chromatography, gel electrophoresis s

What is the first step needed for protein separatiaon

Isolation of protein by centrifugación

How does centrifugation work

Cells are suspended in a liquid medium and are then subjected to high g forces which separate the cell components based on density after they have been homogenised

Centrifugal force

A force appearing to act on an object when viewed in a rotating frame of reference, drenched away from axis rotation

What 2 forces acts upon samples during centrifugation

Buoyant forces + centrifugal forces

Buoyant force

force from the viscosity of the liquid,pushes moelcuels up

Centrifugal force

Forces particle to the bottom of the tube

What si teh general rule for speeds of centrifugation

The large the obj T the quicker it sediments in a centrifuge

Step 2 - How to prepare soluble proteins from cells (6 steps)

Grow in a liquid growth medium, centrifuge at low speeds to form pellet cells, retain the pellet, break open the cells by homogenisation/ sonicaction, centrifuge again at higher speeds to segregate dna and pellet insoluble material,keep the supernatant which has protein of interest in

How does sonicaction work and why is it beneficial

Uses high energy sound waves to pop cells- keep cells cool to prevent denaturation of protein

In biological chromatography what s the stationary pahse often

Some kind of polymer bead which is often functionalists

In biological chromatography what s the Mobile pahse

Is a biologically compatible buffer system specific to the protein/ experiment being performed

How does chromatogrpahy work inn general

Protein mixture in buffer is added to chromatography column filled with stationary phase and buffer, some protein interacts with station pahse so moves slower, other redímanos in mobile pahse so moves more quickly and exists the cholumn first

2 types of chromatography

TLC, column

What can be used to detect proteins as they have eluted from the column is they have no colour

Absorbance at 280n gets plotted as a chromatography

Elution volume

The volyme fo mobile base added before protein comes off the column

How to calculate protein concentraiton of protein from a chromatography

Integrate the area under peak curve

What can chromatography

Interaction with small moelcuels,charge, size, sace, hydrophobicity

Eluate

The product of chromatograms

What can change the elution time

Size exclusion- elation tie is proportional to volume run, changing the properties of buffer systems can chagne elution time

Size exclusion/ gel filtration chromatography

Separates moelcuels based on size/ hydro h dynamic radius → large moelcuels can’t interact with the beads so pass straight through, smaller moelcuels take different apathy’s different paths through the media so separation occur s

What does ion exchange chromatography seapret based off

Charge- overall charge, charge distribution, charge density