Chemistry 4010 Lab It’s all about PROTEINS… It’s all about PROTEINS…

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Text of Chemistry 4010 Lab It’s all about PROTEINS… It’s all about PROTEINS…

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  • Chemistry 4010 Lab Its all about PROTEINS Its all about PROTEINS
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  • SDS-PAGE Understanding SDS and PAGE Understanding SDS and PAGE SDS- Sodium Dodecyl Sulfate a detergent soap that dissolves hydrophobic molecules a detergent soap that dissolves hydrophobic molecules also has a negative charge also has a negative charge by incubating the cell with SDS, the membranes are dissolved and proteins are solubilized by incubating the cell with SDS, the membranes are dissolved and proteins are solubilized protein now possesses negative charges protein now possesses negative charges PAGE- Polyacrylamide Gel Electrophoresis
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  • Understanding SDS and PAGE Understanding SDS and PAGE SDS- Sodium Dodecyl Sulfate PAGE- Polyacrylamide Gel Electrophoresis polymer of acrylamide monomers polymer of acrylamide monomers with a mixture of other buffers and solutions this polymer can be changed into a gel matrix with a mixture of other buffers and solutions this polymer can be changed into a gel matrix electricity is used to pull the protein through the gel electricity is used to pull the protein through the gel the negatively charged protein moves to the positive poles the negatively charged protein moves to the positive poles SDS-PAGE
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  • Side view
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  • Top View
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  • How does it all work add the proteins to the gel matrix add the proteins to the gel matrix turn on the current turn on the current negatively charged proteins will move through the gel negatively charged proteins will move through the gel smaller sized proteins will move at a faster rate due to their ability to maneuver through the gel smaller sized proteins will move at a faster rate due to their ability to maneuver through the gel
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  • Proteins Moving through the Gel
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  • What will the gel look like smaller proteins will move through the gel faster while larger proteins move at a slower pace smaller proteins will move through the gel faster while larger proteins move at a slower pace Visualize the bands by staining the gel with Coomassie Blue Visualize the bands by staining the gel with Coomassie Blue
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  • SDS- PAGE Setup
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  • SDS-PAGE vs. Gel Electrophoresis SDS-PAGE is a great tool for analyzing proteins, whereas Gel Electrophoresis is used to study DNA. SDS-PAGE is a great tool for analyzing proteins, whereas Gel Electrophoresis is used to study DNA.
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  • Gel Electrophoresis Makes use of an agarose gel Makes use of an agarose gel Agarose is a linear polysaccharide (average molecular mass about 12,000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6- anhydrogalactose. Agarose is usually used at concentrations between 1% and 3%.
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  • Gel Electrophoresis DNA is cut by restriction enzymes to yield many different sized pieces of DNA DNA is cut by restriction enzymes to yield many different sized pieces of DNA Like SDS-PAGE, an electric current is applied and the negatively charged DNA moves through the gel Like SDS-PAGE, an electric current is applied and the negatively charged DNA moves through the gel Smaller pieces move faster Smaller pieces move faster
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  • Gel Electrophoresis Setup
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  • THEORY SDS-PAGE & Gel Electrophoresis These techniques make use of the fact that molecules are being separated based on size and charge, based on the following equations These techniques make use of the fact that molecules are being separated based on size and charge, based on the following equations v = the rate (velocity) of migration v = the rate (velocity) of migration E = the strength of the electrical field E = the strength of the electrical field z = the charge on the molecule z = the charge on the molecule f = the frictional force on the molecule f = the frictional force on the molecule Frictional force can then be defined as Frictional force can then be defined as is the viscosity of the medium is the viscosity of the medium r is the stokes radius of the molecule r is the stokes radius of the molecule
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  • Gel Filtration or GPC Gel filtration chromatography is a separation based on size Gel filtration chromatography is a separation based on size stationary phase consists of porous beads with a well- defined range of pore sizes mobile phase consists of the solvent
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  • GPC Proteins that are too large to fit inside any of the pores-EXCLUDED Proteins that are too large to fit inside any of the pores-EXCLUDED access only to the mobile phase between the beads and elute first. Proteins of intermediate size PARTIALLY INCLUDED Proteins of intermediate size PARTIALLY INCLUDED fit inside some but not all of the pores in the beads. These proteins will then elute between the large ("excluded") and small ("totally included") proteins Proteins that are small enough can fit inside all the pores in the beads -INCLUDED Proteins that are small enough can fit inside all the pores in the beads -INCLUDED access to the mobile phase inside the beads as well as the mobile phase between beads and elute last
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  • Theory of GPC These separations can be described by this equation These separations can be described by this equation Vr = the retention volume of the protein Vo = the volume of mobile phase between the beads of the stationary phase inside the column (sometimes called the void volume) Vi = the volume of mobile phase inside the porous beads (also called the included volume) K = the partition coefficient (the extent to which the protein can penetrate the pores in the stationary phase, with values ranging between 0 and 1).
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  • GPC Setup
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  • Conclusion All of these techniques serve to enhance our ability to better elucidate the size, charge and polarity of polymers in science All of these techniques serve to enhance our ability to better elucidate the size, charge and polarity of polymers in science Understanding the parallel nature of these experiments will allow us to bridge the gap between natural and synthetic polymers Understanding the parallel nature of these experiments will allow us to bridge the gap between natural and synthetic polymers