ElectrophoresisElectrophoresis

ElectrophoresisElectrophoresis

Electrophoresis is a commonly used technique Electrophoresis is a commonly used technique to separate proteins, lipoproteins, nucleic acid, to separate proteins, lipoproteins, nucleic acid, particles, emulsion grains, or even bacteria on particles, emulsion grains, or even bacteria on the basis of their net charge in specified the basis of their net charge in specified buffered media.buffered media.

Secara Istilah:Secara Istilah:Pergerakan molekul - molekul yang bermuatan Pergerakan molekul - molekul yang bermuatan dibawah pengaruh medan listrikdibawah pengaruh medan listrik

Principles of SeparationPrinciples of Separation : :

1.1. Charge on the ProteinsCharge on the Proteins

At the pI of a specific protein, the protein At the pI of a specific protein, the protein molecule carries no net charge molecule carries no net charge   and does not migrate in an electric field.   and does not migrate in an electric field. - At pH above the pI, the protein has a net - At pH above the pI, the protein has a net negative charge and migrates negative charge and migrates   towards the anode.   towards the anode. - At pH below the pI, the protein obtains a net - At pH below the pI, the protein obtains a net positive charge on its positive charge on its   surface and migrates towards the cathode.  surface and migrates towards the cathode.

2. 2. Shape of the ProteinsShape of the Proteins

3. 3. Size of the ProteinsSize of the Proteins

SDS-PAGE SDS-PAGE

SDS PAGE SDS PAGE enables separation of proteins by sizeenables separation of proteins by size

SDSSDS

If we want to separate many If we want to separate many different protein molecules different protein molecules of a variety of shapes and of a variety of shapes and sizes, we first want to get sizes, we first want to get them to be linear so that the them to be linear so that the proteins no longer have any proteins no longer have any secondary, tertiary or secondary, tertiary or quaternary structure (i.e. we quaternary structure (i.e. we want them to have the same want them to have the same linear shape). linear shape).

Analogy:Analogy:

Consider two proteins that are each 500 amino acids Consider two proteins that are each 500 amino acids long but one is shaped like a closed umbrella while long but one is shaped like a closed umbrella while the other one looks like an open umbrella. If you tried the other one looks like an open umbrella. If you tried to run down the street with both of these molecules to run down the street with both of these molecules under your arms, which one would be more likely to under your arms, which one would be more likely to slow you down, even though they weigh exactly the slow you down, even though they weigh exactly the same? This analogy helps point out that not only the same? This analogy helps point out that not only the mass but also the shape of an object will determine mass but also the shape of an object will determine how well it can move through and environment. So how well it can move through and environment. So we need a way to convert all proteins to the same we need a way to convert all proteins to the same shape - we use SDS. shape - we use SDS.

CellCell

Incubated with SDSIncubated with SDS

membranes will be dissolved, the proteins will be membranes will be dissolved, the proteins will be soluablized, all the proteins will be covered with soluablized, all the proteins will be covered with

many negative charges many negative charges

- all proteins contain only primary structure - all proteins contain only primary structure - all proteins have a large negative charge which means - all proteins have a large negative charge which means

they will all migrate towards the positve pole when they will all migrate towards the positve pole when placed in an electric field placed in an electric field

SDSSDS Sodium Dodecyl Sulphate (SDS) is an anionic Sodium Dodecyl Sulphate (SDS) is an anionic

detergent that denatures proteins by wrapping the detergent that denatures proteins by wrapping the hydrophobic tail around the polypeptide backbone.hydrophobic tail around the polypeptide backbone.

For almost all proteins, SDS binds at a ratio of For almost all proteins, SDS binds at a ratio of approximately 1.4 g SDS per gram of protein, thus approximately 1.4 g SDS per gram of protein, thus conferring a net negative charge to the polypeptide in conferring a net negative charge to the polypeptide in proportion to its length. proportion to its length.

The SDS also disrupts hydrogen bonds, blocks The SDS also disrupts hydrogen bonds, blocks hydrophobic interactions, and substantially unfolds hydrophobic interactions, and substantially unfolds the protein molecules, minimizing differences in the protein molecules, minimizing differences in molecular form by eliminating the tertiary and molecular form by eliminating the tertiary and secondary structures. secondary structures.

· SDS are attached to the protein · SDS are attached to the protein in a constant ratio, Proteins now in a constant ratio, Proteins now have identical charge density. have identical charge density.

PAGEPAGE If the proteins are denatured and put into an electric If the proteins are denatured and put into an electric

field, they will all move towards the positive pole at field, they will all move towards the positive pole at the same rate, with no separation by size. So we need the same rate, with no separation by size. So we need to put the proteins into an environment that will allow to put the proteins into an environment that will allow different sized proteins to move at different rates. The different sized proteins to move at different rates. The environment of choice is polyacrylamide, which is a environment of choice is polyacrylamide, which is a polymer of acrylamide monomers. When this polymer of acrylamide monomers. When this polymer is formed, it turns into a gel and we will use polymer is formed, it turns into a gel and we will use electricity to pull the proteins through the gel so the electricity to pull the proteins through the gel so the entire process is called polyacrylamide gel entire process is called polyacrylamide gel electrophoresis (electrophoresis (PAGEPAGE). ).

PAGE PAGE

This cartoon shows a slab of polyacrylamide (dark gray) with tunnels This cartoon shows a slab of polyacrylamide (dark gray) with tunnels (different sized red rings with shading to depict depth) exposed on the (different sized red rings with shading to depict depth) exposed on the

edge. Notice that there are many different sizes of tunnels scattered edge. Notice that there are many different sizes of tunnels scattered

randomly throughout the gel.randomly throughout the gel.

Analogy:Analogy:Think of the gel as a tiny forrest with many Think of the gel as a tiny forrest with many branches and twigs througout the forrest but branches and twigs througout the forrest but they form tunnels of different sizes. If we let they form tunnels of different sizes. If we let children and adults run through this forrest at children and adults run through this forrest at the same time, who will be able to get through the same time, who will be able to get through faster? The children of course. Why? Because faster? The children of course. Why? Because of their small size, they are more easily able to of their small size, they are more easily able to move through the forrest. Likewize, small move through the forrest. Likewize, small molecules can manuver through the molecules can manuver through the polyacrylamide forrest faster than big polyacrylamide forrest faster than big molecules. molecules.

An electric filed is established with the positive pole An electric filed is established with the positive pole (red plus) at the far end and the negative pole (black (red plus) at the far end and the negative pole (black minus) at the closer end. Since all the proteins have minus) at the closer end. Since all the proteins have strong negative charges, they will all move in the strong negative charges, they will all move in the direction the arrow is pointing (run to red). direction the arrow is pointing (run to red).

There are two types of buffer systems used in protein There are two types of buffer systems used in protein

gel electrophoresis: continuous and discontinuousgel electrophoresis: continuous and discontinuous. .

A continuous system uses only one buffer for A continuous system uses only one buffer for the tanks and the gel. the tanks and the gel.

In a discontinuous system, a nonrestrictive In a discontinuous system, a nonrestrictive large-pore gel called a stacking gel is layered large-pore gel called a stacking gel is layered on top of a separating (resolving) gel. The two on top of a separating (resolving) gel. The two gel layers are each made with a different gel layers are each made with a different buffer, and the tank buffers differ from the gel buffer, and the tank buffers differ from the gel buffers. buffers.

SDS-PAGE has a number of uses, SDS-PAGE has a number of uses, which include:which include:

Establishing protein size Establishing protein size Protein identification Protein identification Determining sample purity Determining sample purity Identifying disulfide bonds Identifying disulfide bonds Quantifying proteins Quantifying proteins Blotting applications Blotting applications

Procedure:Procedure:1.1. The separating gel.The separating gel. Prepare the separating gel solution except ammonium persulfate and Prepare the separating gel solution except ammonium persulfate and

TEMED in a vacuum flask. De-aerate for 1 min. under TEMED in a vacuum flask. De-aerate for 1 min. under vacuum. After adding the ammonium persulfate and TEMED vacuum. After adding the ammonium persulfate and TEMED mix the solution gently. mix the solution gently.

With a pipet fill the separating gel solution between the glass plate With a pipet fill the separating gel solution between the glass plate sandwich along the edge of one of the spacers, until the height sandwich along the edge of one of the spacers, until the height of the solution is 1.0 cm below the comb. Immediately overlay of the solution is 1.0 cm below the comb. Immediately overlay the solution with water saturated 2-butanol or isopropanol to the solution with water saturated 2-butanol or isopropanol to exclude air and to obtain an even interface between the gels. exclude air and to obtain an even interface between the gels. Allow the gel to polymerize for 45 min. The gel is polymerized Allow the gel to polymerize for 45 min. The gel is polymerized when a sharp interface is visible below the overlay. when a sharp interface is visible below the overlay.

separating gelseparating gel 10 %10 % 15 % 15 % rangerange 16-70 kD16-70 kD 12-45 k 12-45 k waterwater 1600 µl1600 µl 939 µl 939 µl 1.5 M Tris pH 8.81.5 M Tris pH 8.8 1000 µl1000 µl 1000 µl 1000 µl 30% Acrylamide30% Acrylamide 1333 µl1333 µl 2000 µl 2000 µl 10% SDS10% SDS 40 µl40 µl 40 µl 40 µl 20% Ammonium 20% Ammonium persulfatepersulfate 14 µl14 µl 14 µl 14 µl TEMEDTEMED 7 µl7 µl 7 µl 7 µl

2. The stacking gel.2. The stacking gel. Prepare the stacking gel just before using the gel, to Prepare the stacking gel just before using the gel, to

maintain the ion discontinuities at the interface maintain the ion discontinuities at the interface between the two gels. Mix the ingredients and de-between the two gels. Mix the ingredients and de-aerate before adding the ammonium persulfate and aerate before adding the ammonium persulfate and TEMED.TEMED.

Remove the solution from the top of the gel, rinse Remove the solution from the top of the gel, rinse with water and dry the area above the gel carefully with water and dry the area above the gel carefully with filter paper. Fill the stacking gel solution on top with filter paper. Fill the stacking gel solution on top of the separating gel. Place the well forming comb in of the separating gel. Place the well forming comb in position, being careful not to trap air bubbles under position, being careful not to trap air bubbles under the teeth. Visible polymerization of the gel should the teeth. Visible polymerization of the gel should occur within 20 min. occur within 20 min.

stacking gelstacking gel

waterwater 1475 µl1475 µl 0.5 M Tris pH 6.80.5 M Tris pH 6.8 625 µl625 µl 30% Acrylamide 30% Acrylamide 335 µl335 µl 10% SDS10% SDS 25 µl25 µl

20% Ammonium persulfate20% Ammonium persulfate 10 µl10 µl TEMEDTEMED 3 µl3 µl

3. Sample Preparation 3. Sample Preparation

2* Sample buffer:2* Sample buffer: 0.5 M Tris pH 6.80.5 M Tris pH 6.8 2.4 ml2.4 ml 10 % (w/v) SDS10 % (w/v) SDS 4.0 ml4.0 ml 0.2 % Bromophenolblue(w/v in water)0.2 % Bromophenolblue(w/v in water) 0.6 ml0.6 ml GlycerolGlycerol 2.0 ml2.0 ml

Prepare the 2* SDS-reducing sample buffer by Prepare the 2* SDS-reducing sample buffer by adding 100 µl 2-mercaptoethanol to each 0.9 adding 100 µl 2-mercaptoethanol to each 0.9 ml 2* Sample buffer. Dilute the sample (10 µl) ml 2* Sample buffer. Dilute the sample (10 µl) with an equal volume of 2* SDS-reducing with an equal volume of 2* SDS-reducing sample buffer. Heat the samples with sample buffer. Heat the samples with cytoplasmic proteins for 3 min at 95°C. cytoplasmic proteins for 3 min at 95°C. Hydrophobic (membrane) proteins are Hydrophobic (membrane) proteins are incubate 15 min at 37°C ( or 10 min at 60°C) incubate 15 min at 37°C ( or 10 min at 60°C) to avoid aggregation. to avoid aggregation.

4. 4. ElectrophoresisElectrophoresis

Dilute the 5* running buffer with 4 volumes of Dilute the 5* running buffer with 4 volumes of water water

5 * Running buffer5 * Running buffer (for 1 liter)(for 1 liter) TrisTris 15 g15 g GlycineGlycine 72 g72 g SDSSDS 5 g5 g

Assemble the electrophoresis cell, remove Assemble the electrophoresis cell, remove tubing, clamps and comb. Fill the upper and tubing, clamps and comb. Fill the upper and lower reservoir with electrode buffer. Load the lower reservoir with electrode buffer. Load the samples into the wells in the stacking gel. samples into the wells in the stacking gel. Connect the electrophoresis unit to the power Connect the electrophoresis unit to the power supply. The lower electrode is the anode (+) supply. The lower electrode is the anode (+) and the upper is the cathode (-). Start with a and the upper is the cathode (-). Start with a low voltage (70 volt) and increase to a higher low voltage (70 volt) and increase to a higher (200 volt) when the samples entered the (200 volt) when the samples entered the stacking gel, continue the electrophoresis until stacking gel, continue the electrophoresis until the blue dye has reached the bottom of the gel. the blue dye has reached the bottom of the gel.

Detection of Proteins in GelsDetection of Proteins in Gels

Dye staining with Coomassie Brilliant Blue Dye staining with Coomassie Brilliant Blue R250R250

Staining solution (5/5/1):Staining solution (5/5/1): Coomassie Brilliant Blue R-250Coomassie Brilliant Blue R-250 0.1 % (w/v)0.1 % (w/v) MethanolMethanol 45.5 % (v/v)45.5 % (v/v) Acetic acidAcetic acid 9.0 % (v/v)9.0 % (v/v)

Filter the solution before use. Soak the gel in Filter the solution before use. Soak the gel in an excess of staining solution for 1 hour. an excess of staining solution for 1 hour. Destain with destain solution, with a Kleenex Destain with destain solution, with a Kleenex Tissue (to absorb the CCB-R250) until the Tissue (to absorb the CCB-R250) until the background is clear. background is clear.

Destain solution:Destain solution: MethanolMethanol 5.0 % (v/v)5.0 % (v/v) Acetic acidAcetic acid 7.0 % (v/v)7.0 % (v/v)

Staining with Coomassie Brilliant Blue G-Staining with Coomassie Brilliant Blue G-250.250.

Fixation for at least 1 h in 12 % (W/V) TCA Fixation for at least 1 h in 12 % (W/V) TCA Staining solution:Staining solution: Coomassie Brilliant BlueCoomassie Brilliant Blue G-250G-250 0.1 % (w/v)0.1 % (w/v) Phosphoric acidPhosphoric acid 2.0 % (w/v)2.0 % (w/v) Ammonium sulfateAmmonium sulfate 6.0 % (w/v)6.0 % (w/v)

The staining solution should be prepared in the The staining solution should be prepared in the following sequence: only after complete following sequence: only after complete solubilization of the ammonium sulfate in the solubilization of the ammonium sulfate in the acid the aliquot volume of a stock solution of acid the aliquot volume of a stock solution of CBB G-250 (1 g/ 20 ml water) should be CBB G-250 (1 g/ 20 ml water) should be added. The staining solution should never be added. The staining solution should never be passed through a filter. The staining solution passed through a filter. The staining solution should be shaken prior to use for even should be shaken prior to use for even distribution of the colloidal particles. distribution of the colloidal particles. Stain until the bands are clearly visible (2 Stain until the bands are clearly visible (2 hours or o/n). Destain with destain solution. hours or o/n). Destain with destain solution.

This shows a top view of an SDS PAGE after the This shows a top view of an SDS PAGE after the

current has been on for a while (positive pole atcurrent has been on for a while (positive pole at the bottom) and then turned off. The gel (gray box) the bottom) and then turned off. The gel (gray box)

has five numbered lanes where five different has five numbered lanes where five different samples of proteins (many copies of each kind of samples of proteins (many copies of each kind of

protein) were applied to the gel. (Lane 1, molecular protein) were applied to the gel. (Lane 1, molecular weight standards of known sizes; Lane 2, a mixture weight standards of known sizes; Lane 2, a mixture of three proteins of different sizes with a being the of three proteins of different sizes with a being the biggest and c being the smallest protein; Lane 3, biggest and c being the smallest protein; Lane 3,

protein a by itself; Lane 4, protein b by itself; Lane protein a by itself; Lane 4, protein b by itself; Lane 5 protein c by itself.) Notice that each group of the 5 protein c by itself.) Notice that each group of the

three proteinsthree proteins migrated the same distance in migrated the same distance in

the gel whether they were with other proteins the gel whether they were with other proteins (lane 2) or not (lanes 3-5). The molecular (lane 2) or not (lanes 3-5). The molecular weight standards are used to measure the weight standards are used to measure the relative sizes of the unknow proteins (a, b, relative sizes of the unknow proteins (a, b,

and c).and c).

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