PROTEOMICS

JEGANATHAN C III-YEAR

DEPARTMENT OF BIOMEDICAL SCIENCE

ULTRAFILTERATION

• Ultrafilteration ,also referred to as reverse osmosis , is a process in which solvents ,up to a certain critical size, are forced through a barrier membrane by a higher pressure on one side of the membrane than the other

• This technique is primarily used for concentrating proteins in dilute solutions .it can be used as a purification step, but it only provides two fractions : bigger molecules found in the retentate, and smaller molecules passing through the membrane and found in ultrafilterate.

CONTINUOUS…

• Ultrafiltration membranes come in a wide range of pore sizes;

• To the pore size of an ultrafiltration membrane ,it is more common to use a nominal molecular weight cutoff (NMWC) for the membrane

• NMWC is defined as the minimum molecular weight globular protein which will not pass through the membrane

PRECIPITATION

• Precipitation is widely used for the recovery of biomolecules

• Induced by: a salt an onganic solvent by changing the pH to alter the nature of

the solution

PRECIPITATION WITH ACETONE

• Many proteins can be precipitated by addition of water-miscible organic solvents such as ethanol and acetone

• Addition of ethanol or acetone to an aqueous protein extract will lead to protein precipitation due to a reduction in water activity

Continuous…

• As the concentration of the organic solvent increases the dissolvent power of water for a charged , hydrophilic protein decreases

• Some hydrophobic , membrane bound proteins are not precipitated by organic solvents but are rather solubilized from the membrane by organic solvents as the solvent displaces the water molecules from around the hydrophobic patches of the protein

• In water – solvent mixtures ,the interactions of water with proteins is reduced because water becomes ordered around solvent molecules.

• Organic solvent precipitation is routinely carried out at 0 degree Celsius and can be incubated at subzero temperatures since the miscible solvents form mixtures that freeze well below 0 degree Celsius.

• The addition of the organic solvents causes heat evolution so care should be taken to keep the temperature at or below zero

• The first addition should be performed slowly with efficient cooling .

• The protein concentration can be 5-30mg/ml but the salt concentration should be around 0.05-0.2M.

MATERIALS

• Most proteins will precipitate in the range of 20-50% V/V acetone .

• Add acetone to the protein solution to reach the desired percentage and incubated at 0 degree Celsius for 15 min

• Collect the precipitated material by centrifugation in pre-cooled rotor at 3000xg for 10 min. save the supernatant .

• Redisolve the precipitated in a minimal volume of cold buffer .do not add excess buffer. If the precipitate does not dissolve , there is probably denatured protein present.

• At 50% solvent concentration ,only proteins of molecular weight less than 15,000Da are likely to remain solution

• The starting protein concentration should be >1mg/1ml

PRECIPITATION WITH POLYETHYLENE GLYCOL (PEG)

• The use of nonionic polymers for protein purification deserves wider application

• PEG is not easy to remove from a protein fraction , low levels of PEG are compatible with many procedures

MATERIALS

• PEG-6000:50% (w/v) PEG-6000 in distilled water warmed to 37 degree Celsius to speed up the solubilization

• Phosphate buffer: 55mM potassium phosphate buffer , pH 7.0

carry out all addition at 4 degree celsius with stirring. The protein solution , add PEG solution to a final concentration of 35% (w/v) and stir for 30min

Let the suspension stand for 5min then centrifuge for 30min at 35,000 x g at 4 degree Celsius

Discard the supernatant and dissolve the pellet in 10ml of phosphate buffer .centrifuge for 30min at 35,000 x g at 4 degree Celsius. Discard the pellet . The supernatant should be clear

Add the PEG solution to supernatant to a concentration of 2.5% PEG. Allow the suspension to stand for 5min, then centrifuge for 30min at 35,000x g at 4 degree Celsius .

The precipitate represents fraction1. resuspend the precipitate in desired buffer

Prepare fractions of 4.5%, 6.5%, 8.5%, 10.0%, and 14.5% PEG.

RECOVERY OF PROTEIN BY TCA PRECIPITATION

• Using 100% (w/v) TCA stock solution adjust the volume of the protein solution to a final concentrations of 10%(v/v) TCA and incubate on ice for 30min. Centrifuge at 12,000x g for 5min

• Discard the supernatant .to remove the TCA, rinse the pellet with ethanol: ether (1:1 v/v) then centrifuge at 12,000x g for 5min

• Dissolve the protein precipitate in 1 x SDS sample buffer and boil for 5min. The sample should be blue in color. If it is yellow , then the pH is separation. Adjust the pH by adding microliter amounts of either unbuffered Tris or NH4OH until the sample turns blue

• Analyze the sample by SDS-PAGE. Alternatively the boiled samples can be stored at -20 degree celsius

RECOVERY OF PROTEIN FROM DILUTE SOLUTIONS BY METHANOL CHLOROFORM PRECIPITATION

• Add 0.4ml of methanol to 0.1ml of protein solution

• Vortex mix, then centrifuge for 10sec at 12,000 x g

• Add 0.1ml of chloroform(0.2ml when the sample contains a high concentration of phospholipid) ,vortex mix and centrifuge at 12,000 x g for sec.

• Add 0.3ml of water , vortex mix and centrifuge 1min at 12,000 x g.

• Remove upper layer and discard• Add 0.3mi of methanol to lower

phase(chloroform phase). Mix and centrifuge 2min at 12,000 x g.

• Remove the supernatant an dry the protein pellet under a stream of nitrogen