Transcript

Chemistry 4010 Chemistry 4010 LabLab It’s all It’s all

about about PROTEINSPROTEINS……

SDS-PAGESDS-PAGE

Understanding SDS and PAGEUnderstanding SDS and PAGE– SDS- Sodium Dodecyl SulfateSDS- Sodium Dodecyl Sulfate

a detergent soap that dissolves a detergent soap that dissolves hydrophobic moleculeshydrophobic molecules

also has a negative chargealso has a negative charge by incubating the cell with SDS, the by incubating the cell with SDS, the

membranes are dissolved and proteins are membranes are dissolved and proteins are solubilizedsolubilized

protein now possesses negative chargesprotein now possesses negative charges

– PAGE- Polyacrylamide Gel PAGE- Polyacrylamide Gel ElectrophoresisElectrophoresis

Understanding SDS and PAGEUnderstanding SDS and PAGE– SDS- Sodium Dodecyl SulfateSDS- Sodium Dodecyl Sulfate– PAGE- Polyacrylamide Gel ElectrophoresisPAGE- Polyacrylamide Gel Electrophoresis

polymer of acrylamide monomerspolymer of acrylamide monomers with a mixture of other buffers and solutions with a mixture of other buffers and solutions

this polymer can be changed into a gel matrixthis polymer can be changed into a gel matrix electricity is used to pull the protein through electricity is used to pull the protein through

the gelthe gel the negatively charged protein moves to the the negatively charged protein moves to the

positive polespositive poles

SDS-PAGESDS-PAGE

Side view Side view

Top ViewTop View

How does it all workHow does it all work

add the proteins to the gel matrixadd the proteins to the gel matrix turn on the currentturn on the current negatively charged proteins will negatively charged proteins will

move through the gelmove through the gel smaller sized proteins will move smaller sized proteins will move

at a faster rate due to their ability at a faster rate due to their ability to maneuver through the gelto maneuver through the gel

Proteins Moving Proteins Moving through the Gelthrough the Gel

What will the gel look What will the gel look likelike

smaller proteins smaller proteins will move through will move through the gel faster the gel faster while larger while larger proteins move at proteins move at a slower pacea slower pace

Visualize the Visualize the bands by staining bands by staining the gel with the gel with Coomassie BlueCoomassie Blue

SDS- PAGE SetupSDS- PAGE Setup

SDS-PAGE vs. Gel SDS-PAGE vs. Gel ElectrophoresisElectrophoresis

SDS-PAGE is a SDS-PAGE is a great tool for great tool for analyzing analyzing proteins, proteins, whereas Gel whereas Gel Electrophoresis is Electrophoresis is used to study used to study DNA.DNA.

Gel ElectrophoresisGel Electrophoresis

Makes use of an agarose gelMakes use of an agarose gel– Agarose is a linear polysaccharide Agarose is a linear polysaccharide

(average molecular mass about (average molecular mass about 12,000) made up of the basic repeat 12,000) made up of the basic repeat unit agarobiose, which comprises unit agarobiose, which comprises alternating units of galactose and alternating units of galactose and 3,6-anhydrogalactose. Agarose is 3,6-anhydrogalactose. Agarose is usually used at concentrations usually used at concentrations between 1% and 3%.between 1% and 3%.

Gel ElectrophoresisGel Electrophoresis

DNA is cut by restriction enzymes DNA is cut by restriction enzymes to yield many different sized to yield many different sized pieces of DNApieces of DNA

Like SDS-PAGE, an electric Like SDS-PAGE, an electric current is applied and the current is applied and the negatively charged DNA moves negatively charged DNA moves through the gelthrough the gel

Smaller pieces move fasterSmaller pieces move faster

Gel Electrophoresis Gel Electrophoresis SetupSetup

THEORYTHEORYSDS-PAGE & Gel SDS-PAGE & Gel ElectrophoresisElectrophoresis

These techniques make use of the fact that molecules are These techniques make use of the fact that molecules are being separated based on size and charge, based on the being separated based on size and charge, based on the following equationsfollowing equations

v = the rate (velocity) of migrationv = the rate (velocity) of migration E = the strength of the electrical fieldE = the strength of the electrical field z = the charge on the molecule z = the charge on the molecule f = the frictional force on the moleculef = the frictional force on the molecule

Frictional force can then be defined asFrictional force can then be defined as

ηη is the viscosity of the medium is the viscosity of the medium r is the stokes radius of the moleculer is the stokes radius of the molecule

Gel Filtration or GPCGel Filtration or GPCGel filtration Gel filtration

chromatography is a chromatography is a separation based on sizeseparation based on size–stationary phase consists stationary phase consists of porous beads with a of porous beads with a well-defined range of pore well-defined range of pore sizessizes

–mobile phase consists of mobile phase consists of the solventthe solvent

GPCGPC

Proteins that are too large to fit inside any of Proteins that are too large to fit inside any of the pores-EXCLUDEDthe pores-EXCLUDED– access only to the mobile phase between the access only to the mobile phase between the

beads and elute first.beads and elute first. Proteins of intermediate size –PARTIALLY Proteins of intermediate size –PARTIALLY

INCLUDEDINCLUDED– fit inside some but not all of the pores in the beads. fit inside some but not all of the pores in the beads.

These proteins will then elute between the large These proteins will then elute between the large ("excluded") and small ("totally included") proteins("excluded") and small ("totally included") proteins

Proteins that are small enough can fit inside Proteins that are small enough can fit inside all the pores in the beads -INCLUDEDall the pores in the beads -INCLUDED– access to the mobile phase inside the beads as well access to the mobile phase inside the beads as well

as the mobile phase between beads and elute lastas the mobile phase between beads and elute last

Theory of GPCTheory of GPC

These separations can be described by These separations can be described by this equation this equation – Vr = the retention volume of the proteinVr = the retention volume of the protein– Vo = the volume of mobile phase between Vo = the volume of mobile phase between

the beads of the stationary phase inside the the beads of the stationary phase inside the column (sometimes called the void volume)column (sometimes called the void volume)

– Vi = the volume of mobile phase inside the Vi = the volume of mobile phase inside the porous beads (also called the included porous beads (also called the included volume)volume)

– K = the partition coefficient (the extent to K = the partition coefficient (the extent to which the protein can penetrate the pores in which the protein can penetrate the pores in the stationary phase, with values ranging the stationary phase, with values ranging between 0 and 1). between 0 and 1).

GPC SetupGPC Setup

ConclusionConclusion

All of these techniques serve to All of these techniques serve to enhance our ability to better enhance our ability to better elucidate the size, charge and elucidate the size, charge and polarity of polymers in sciencepolarity of polymers in science

Understanding the parallel nature Understanding the parallel nature of these experiments will allow us of these experiments will allow us to bridge the gap between natural to bridge the gap between natural and synthetic polymersand synthetic polymers


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