SDS-PAGE, Sodium Dodecyl Sulfate–PolyAcrylamide Gel Electrophoresis–Animation

Summary

SDS-PAGE is a powerful technique for protein analysis, separating proteins based on molecular weight using polyacrylamide gel electrophoresis. Proteins are denatured and bound with SDS, then loaded into a gel system with a stacking and separating gel. An electric field drives negatively charged proteins through the gel, allowing size-based separation. The process involves sample preparation, gel polymerization, and electrophoresis, culminating in protein bands that can be visualized and analyzed using staining and immunological methods.

Takeaways

• 🧪 SDS-PAGE is a powerful technique for studying proteins, separating them based on molecular weight using polyacrylamide gel electrophoresis.
• 🔬 Sample preparation involves adding a loading buffer with SDS, beta-mercaptoethanol, bromophenol blue, and glycerol to protein samples, which may come from various biological or environmental sources.
• 🔥 Heating samples at 95°C for five minutes helps denature proteins, disrupting their natural structure and interactions.
• 🌊 SDS is an anionic surfactant that binds to proteins, neutralizing their intrinsic charges and allowing separation based on molecular weight alone.
• 🔗 Amino acids, the building blocks of proteins, are linked by peptide bonds and fold into specific spatial conformations influenced by various interactions.
• 🌡 The gel setup includes a separating gel with pH 8.8 and a stacking gel with pH 6.8, both made of acrylamide and other components to create a porous structure.
• 🚀 The gel polymerization process is initiated by ammonium persulfate and accelerated by TEMED, resulting in a gel with specific porosity for protein separation.
• 🏎️ The stacking gel ensures that proteins enter the separating gel simultaneously, preventing smearing and improving separation clarity.
• 🌀 The running buffer with glycine and chloride ions, along with SDS, maintains the protein charge state during electrophoresis.
• 📊 The electrophoresis process sorts proteins by size, with smaller proteins migrating more easily through the gel matrix than larger ones.
• 🖼️ After separation, proteins can be visualized and analyzed using staining methods like Coomassie blue, which binds to proteins and creates visible bands.

Q & A

What is SDS-PAGE and how is it used in protein studies?

SDS-PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a method used to separate proteins based on their molecular weight. It involves the use of a polyacrylamide gel and is a powerful technique in the study of proteins.

What is the purpose of adding a loading buffer to protein samples in SDS-PAGE?

The loading buffer, containing SDS, beta-mercaptoethanol, bromophenol blue, and glycerol, is added to denature proteins, break disulfide bonds, visualize the sample, and increase sample density to ensure it falls to the bottom of the well during electrophoresis.

Why are proteins heated at 95 degrees Celsius before being loaded onto the gel?

Heating the proteins at 95 degrees Celsius helps to denature them, ensuring that they are in a linear form and that their intrinsic charges are negligible compared to the negative charges from SDS, which is necessary for uniform separation based on molecular weight.

What is the role of SDS in the SDS-PAGE process?

SDS, an anionic surfactant, denatures the native proteins by disrupting hydrogen, hydrophobic, and ionic interactions. It binds uniformly to the proteins, giving them a net negative charge, which allows for separation based on molecular weight.

How are the separating and stacking gels prepared for SDS-PAGE?

The separating gel solution has a pH of 8.8, and the stacking gel solution has a pH of 6.8. Both are prepared with acrylamide, ammonium persulfate, and TEMED. The polymerization process involves a free radical initiator and accelerator, resulting in a gel with characteristic porosity.

What is the significance of the pH difference between the separating and stacking gels?

The pH difference between the separating gel (pH 8.8) and the stacking gel (pH 6.8) is crucial for the migration of proteins. The lower pH in the stacking gel slows down the migration of charged molecules, allowing proteins to concentrate into a narrow zone before entering the separating gel.

Why is a molecular weight size marker loaded onto the gel along with the samples?

A molecular weight size marker, consisting of proteins of known sizes, is loaded to provide a reference for estimating the sizes of the proteins in the actual samples. This allows for the determination of the molecular mass of unknown proteins.

How does the electric field affect the migration of proteins in the gel?

The electric field causes the negatively charged proteins to migrate towards the positive electrode. Smaller proteins can move more easily through the gel matrix, while larger proteins are retained and migrate more slowly, leading to separation based on size.

What is the function of the running buffer in SDS-PAGE?

The running buffer, containing glycine and chloride ions, is used to maintain the pH and ionic strength during electrophoresis. It also ensures that the proteins remain denatured and that the pH conditions are suitable for the migration of proteins and ions.

What happens to the proteins when they enter the separating gel from the stacking gel?

Upon entering the separating gel, the pH changes to 8.8, causing glycine to become negatively charged and migrate faster than the proteins. This results in the separation of proteins based on their molecular weight, with higher molecular weight proteins moving more slowly.

How can the separated proteins be analyzed after electrophoresis?

After electrophoresis, proteins can be analyzed using staining methods, such as Coomassie blue, to visualize the protein bands. Additionally, techniques like Western blot can be used for immunological detection of specific proteins.

Outlines

00:00

🔬 SDS-PAGE Technique and Sample Preparation

The paragraph introduces the SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) method, a crucial technique in protein analysis. It explains how proteins, once denatured and combined with SDS, are separated based on their molecular weight in a polyacrylamide gel. The process involves sample preparation with a loading buffer containing SDS, beta-mercaptoethanol, bromophenol blue, and glycerol, followed by heating at 95°C. The paragraph details the denaturation of proteins by SDS, which disrupts the protein's structure and allows separation based on size alone. It also describes the creation of the gel matrix, involving a separating gel with pH 8.8 and a stacking gel with pH 6.8, both composed of acrylamide, ammonium persulfate, and TEMED. The gel's porosity is determined by the ratio of acrylamide to Biss, with different concentrations suitable for various molecular weights. The application of an electric field initiates the migration of negatively charged proteins towards the positive electrode, with smaller proteins moving more easily through the gel matrix.

05:02

🚀 Mechanism of Protein Separation and Staining in SDS-PAGE

This paragraph delves into the mechanism of protein separation in the SDS-PAGE process. It explains the role of the stacking gel in ensuring proteins enter the separating gel simultaneously, which is crucial for obtaining sharp bands. The running buffer's composition, consisting of glycine and chloride ions, is highlighted, with glycine's charge state varying based on pH. At the running buffer's pH of 8.3, glycine is negatively charged, while at the stacking gel's pH of 6.8, it becomes predominantly neutral, slowing down the migration of proteins and chloride ions. This concentration of proteins into a narrow zone facilitates their separation in the separating gel, where pH 8.8 allows glycine to migrate faster than proteins, leading to their size-based separation. The paragraph concludes with the description of protein detection post-electrophoresis, mentioning the use of Coomassie blue as a staining agent to visualize proteins as blue bands against a clear background. Molecular weight markers are used to estimate the size of unknown proteins by comparing their migration distance relative to the markers.

Keywords

💡SDS-PAGE

SDS-PAGE stands for Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, a widely used technique in biochemistry for the separation of proteins based on their molecular weight. In the script, it is the central technique being discussed, with a detailed explanation of how proteins are denatured and separated within a polyacrylamide gel.

💡Proteins

Proteins are large biomolecules composed of amino acid residues linked by peptide bonds. They play a crucial role in the video script as the subject of study in SDS-PAGE, where their molecular weights determine their migration speed through the gel matrix.

💡Molecular Weight

Molecular weight refers to the mass of a molecule, and in the context of the video, it is the key factor in how proteins are separated during SDS-PAGE. Smaller proteins move more easily through the gel's pores, while larger ones are slower, allowing for size-based separation.

💡Acrylamide

Acrylamide is a monomer used in the formation of polyacrylamide gels, which serve as the medium in which proteins are separated during SDS-PAGE. The script describes how acrylamide is polymerized to create a gel with specific porosity that affects the migration of proteins.

💡Beta-Mercaptoethanol

Beta-Mercaptoethanol is a reducing agent used in sample preparation for SDS-PAGE. It is mentioned in the script as a component that cleaves disulfide bonds in proteins, which is essential for their denaturation and subsequent separation by molecular weight.

💡Peptide Bonds

Peptide bonds are the covalent bonds that link amino acids together to form proteins. The script explains that proteins are formed by linking amino acids with peptide bonds, which is fundamental to their structure and function.

💡Denaturation

Denaturation is the process by which proteins lose their native structure, often due to the application of heat or chemical agents. In the script, SDS is described as an agent that denatures proteins, allowing them to be separated based solely on molecular weight.

💡Polymerization

Polymerization is the process of forming polymers from monomers. The script describes how acrylamide monomers undergo a vinyl addition polymerization reaction to form the polyacrylamide gel used in SDS-PAGE.

💡Stacking Gel

The stacking gel is a component of the gel electrophoresis system described in the script. It has a different pH and porosity compared to the separating gel, and it is used to concentrate the proteins into a narrow zone before they enter the separating gel.

💡Running Buffer

The running buffer is the electrolyte solution that facilitates the movement of charged particles through the gel during electrophoresis. The script explains that the pH of the running buffer, along with the presence of SDS, ensures that proteins remain denatured and move based on their charge and size.

💡Coomassie Blue

Coomassie Blue is a protein stain mentioned in the script for visualizing proteins after electrophoresis. It binds specifically to proteins, allowing them to be seen as blue bands against a clear background, which is essential for analyzing the results of an SDS-PAGE run.

Highlights

SDS-PAGE is a powerful technique for studying proteins, separating them based on molecular weight using polyacrylamide gel electrophoresis.

Sample preparation involves adding a loading buffer containing SDS, beta-mercaptoethanol, bromophenol blue, and glycerol to protein samples.

Proteins are heated at 95 degrees Celsius to denature them, facilitating uniform SDS binding and charge distribution.

Proteins are large biomolecules composed of amino acid residues linked by peptide bonds, folding into specific spatial conformations influenced by various interactions.

SDS is an anionic surfactant that denatures proteins by disrupting hydrogen bonds, hydrophobic interactions, and ionic bonds.

Beta-mercaptoethanol acts as a reducing agent to cleave disulfide bonds in proteins.

The polyacrylamide gel's pore size is determined by the ratio of acrylamide to bis-acrylamide, with low concentrations preferred for high molecular weight samples.

The gel is created through a polymerization process initiated by ammonium persulfate and accelerated by TEMED.

A separating gel with pH 8.8 and a stacking gel with pH 6.8 are used, both contributing to the gel's characteristic porosity.

The stacking gel ensures that all proteins enter the separating gel simultaneously, preventing smeared bands.

The running buffer, containing glycine and chloride ions, maintains the proteins' denatured state throughout the electrophoresis process.

Molecular weight markers are used alongside samples to estimate the sizes of proteins in the samples.

Bromophenol blue in the samples helps visualize the sample in the well, while glycerol increases sample density for proper well placement.

The application of an electric field causes negatively charged proteins to migrate towards the positive electrode.

Smaller proteins migrate more easily through the gel matrix, while larger proteins are retained and migrate slower.

The pH of the running buffer and the gel affects the charge state of glycine, influencing protein migration speed.

Proteins are separated by size in the separating gel, with Coomassie blue being a popular staining method for visualization.

After electrophoretic separation, proteins can be analyzed using other methods such as Western blot for immunological detection.

The separating gel can be stained and the molecular weight of unknown proteins determined by comparing to size markers.

Transcripts

sds-page your sodium dodecyl sulfate

polyacrylamide gel electrophoresis is an

electrophoresis method and very powerful

technique in the study of proteins an

sds-page proteins are separated in a

poly acrylamide gel based on their

molecular weight for sample preparation

a loading buffer containing SDS beta

mercaptoethanol bromophenol blue and

glycerol is added to the protein samples

protein samples maybe biologically

derived for example from prokaryotic or

you create excels tissues viruses

environmental samples or purified

proteins when the sample buffer has been

added the samples are then heated at 95

degrees Celsius for five minutes

proteins are large biomolecules

consisting of one or more long chains of

amino acid residues

Meno acids are organic compounds that

contain a mined and carboxyl functional

groups along with the sidechain proteins

are formed by linking amino acids with

peptide bonds and fold into one or more

specific spatial confirmations driven by

a number of interactions such as

hydrogen bonds hydrophobic interaction

disulfide bonds and ionic bonds SDS is

an anionic surfactant and contain a

polar head group with a net negative

charge at the end of a long hydrophobic

carbon chain SDS denatures the native

proteins by disturbing the hydrogen

bonds hydrophobic and ionic interactions

while the reducing agent beta

mercaptoethanol is used to cleave the

disulfide bonds

also when a protein mixture is heated

SDS binds uniformly to the protein and

the intrinsic charges of this protein

become negligible when compared to the

negative charges contributed by SDS this

treatment brings the folded proteins

down to linear molecules with net

negative charge therefore these proteins

can be separated in a poly acrylamide

gel based on their molecular weight

Vergil preparation a separating gel

solution with ph 8.8 and a stacking gel

solution with ph 6.8 are used and both

consist of acrylamide this acrylamide

ammonium persulfate and tea med the gel

is produced by polymerization between

two glass plates anchored vertically in

a cassette the separating gel is poured

first

the polymerization reaction is a vinyl

addition polymerization initiated by a

free radical generating system ammonium

persulfate is used as the free radical

initiator while tea med accelerates the

rate of formation of free radicals from

persulfate these free radicals convert

acrylamide monomers to free radicals

which react with unactivated monomers to

begin the polymerization chain reaction

the elongating polymer chains are

randomly cross-linked by Biss acrylamide

resulting in a gel with a characteristic

porosity

the pore size of the gel is related to

the ratio of acrylamide to Biss

acrylamide low concentration is

preferred for high molecular weight

samples the stacking gel solution is

poured on top of the solid separating

gel

and a plastic comb placed on the top of

the stacking gel during polymerization

enables the formation of small wells in

the gel once the gel completely

polymerizes the comb is removed and the

gel cassette is placed vertically

between two electrodes positive

electrode located at the bottom of the

gel whereas negative electrode

positioned at the top of the gel

the gel is inserted into a chamber

and at risk lysine chloride buffer

system with ph 8.3 is poured to allow

the conduction of current through the

gel SDS is also present in the gel and

in the running buffer to make sure that

once the proteins are denatured they

stay that way throughout the run

for sample application in addition to

the samples a molecular weight size

marker is usually loaded onto the gel

this consists of proteins of known sizes

and thereby allows the estimation of the

sizes of the proteins in the actual

samples which migrate in parallel in

different tracks of the gel each sample

is pivoted into its own well in the gel

and as we have seen previously Roma

phenol blue and glycerol are present in

the samples bromophenol blue is a dye

that is useful for visualizing the

sample in the well and glycerol

increases the density of a sample and

used to make the sample fall to the

bottom of the sample well rather than

just flow out and mix with the running

buffer

after the sample application procedure

an electric field is applied across the

gel

causing the negatively charged proteins

to migrate across the gel away from the

negative electrode and towards the

positive electrode

also small proteins migrate relatively

easily through the mesh of the gel while

larger proteins are more likely to be

retained and thereby migrate more slowly

through the gel the stacking gel is used

to ensure that all of the proteins

arrive at the separating gel at the same

time because gel wells are around one

centimeter deep so in the absence of a

stacking gel proteins would all enter

the separating gel at different times

resulting in very smeared bands

as we have seen previously the running

buffer consists of glycine and chloride

ions so in the sample wells we will have

proteins chloride ions and lysine

proteins and chloride ions are

negatively charged

well glycine can exist in three

different charged States depending on

the pH the ice electric pointer pH I of

glycine is equal 5.97 at this pH glycine

carries neutral charge due the gain and

loss of protons at a pH below its pH I

glycine carries a net positive charge

and at a pH above its pH I it becomes

negatively charged

the running buffer pH is equal 8.3 which

above the pH I of glycine so at this pH

glycine is negatively charged once the

electric current is applied proteins

chloride ions and lysine are forced to

enter the stacking gel where the pH is

equal six point eight at this pH glycine

switches predominantly to this material

ik neutrally charged State this loss of

charge causes them to move very slowly

in the electric field the chloride ions

on the other hand move much more quickly

chloride ions migrate in front of the

proteins as leading ions and lysine

molecules migrate behind the proteins as

initial trailing ions consequently

proteins are trapped and concentrated

into a narrow zone between the chloride

ions and glycine this procession carries

on until it hits the separating gel

where the pH switches 28.8 at this pH

the glycine molecules are mostly

negatively charged and can migrate much

faster than the proteins proteins are

separated so higher molecular weight

proteins move more slowly through the

porous acrylamide gel than lower

molecular weight proteins

due to the relatively small molecule

size of Brahma phenyl blue it migrates

faster than proteins and by optical

control of the migrating colored band

the electrophoresis can be stopped

before the dye and before the samples

have completely migrated through the gel

and leave it at the end of the electro

phoretic separation all proteins are

sorted by size and can then be analyzed

by other methods for example protein

staining methods and immunological

detection such as the Western blot

technique after removing the glass

plates the stacking gel is discarded and

the separating gel can be stained

Kumasi blue is the most popular protein

stain it is an anionic dye which nan

specifically binds to proteins the

structure of Kumasi blue is

predominantly nonpolar and it is usually

used in methanol excision acidified with

acetic acid proteins in the gel are

fixed by acetic acid and simultaneously

stained after staining the proteins are

detected as blue bands on a clear

background the molecular weight size

markers in a separate lane in the gel

can be used to determine the approximate

molecular mass of unknown biomolecules

by comparing the distance traveled

relative to the marker

you