SDS-PAGE

SDS-PAGE stands for:

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Amino acid subunits

1. carboxyl group

2. amino group

3. unique R-group

4. alpha-carbon

Amino acids are connected via? How are these bonds made?

• Peptide bonds

• Dehydration condensation reaction wherein there is a loss of water molecule

Amide or amido group is composed of?

peptide bond and amino group of the other amino acid

Describe a notable characteristic of an amide and what this means

• It has a partial double bond character involving a lone pair of electrons/nitrogen that is localized (which means that they can jump around)

• Makes them not as rotatable as single bonds nor rigid as double bonds

Hierarchy of protein structure

1. Primary

2. Secondary

3. Tertiary

4. Quaternary

Describe primary structure

linear sequence of a chain of amino acids

Describe secondary structure

local folding of the polypeptide chains into helices or sheets

Simplest motif of the protein

Secondary structure

Describe tertiary structure

3D folding/aggregation of secondary structures due to side-chain interactions

Describe quaternary structure

Oligomerization state of the proteins wherein monomers are converted to polymers through polymerization

Hemoglobin is described as?

Tetrameric made up of 2 ɑ/alpha chains and 2 β/beta chains

What is the driving force between the assembly of tertiary structures into quaternary?

Noncovalent interactions such as hydrophobic or Van der Waals

What bonds keep quaternary structures intact

Disulfide linkages

The higher the band of the gel ⇒ the ___ the molecular size

larger

The ___ the band of the gel ⇒ the smaller the molecular size

lower

Molecular biology technique that characterizes proteins based on their MW or molecular size

SDS-PAGE

The main information you can derive from SDS-PAGE

number of individual/distinct proteins and their corresponding molecular size/weight

Two types of gel electrophoresis

1. Vertical system

2. Horizontal system

Basic parts of gel electrophoresis

1. Power supply

2. Chamber

3. Gel Wells

4. Buffer

5. Two electrodes (anode and cathode)

Positive anode? Negative anode? Position in SDS-PAGE?

Positive → anode → bottom; negative → cathode → top

Innate nature of protein charges?

They do not have a uniform charge

It's much ___ for proteins to pass through the pores because of its ___

harder, complex structure

Two factors in SDS-PAGE with proteins

1. Multiple subunits (in a quaternary structure, for example) can have different charges

2. In terms of structure, proteins are complex aggregates and folds into different conformations

How to achieve SDS-PAGE with proteins?

subject proteins to a chemical treatment before loading them onto polyacrylamide gel

Two chemicals applied to proteins prior to SDS-PAGE

1. Sodium Dodecyl Sulfate (SDS)

2. 2-mercaptoethanol/β-mercaptoethanol

What does SDS do?

1. Detergent that will denature proteins, making them linear/cylindrical

2. SDS monomers are naturally negatively charged and wrap around the proteins

What does 2-mercaptoethanol do?

Disulfide linkages will be cut/cleaved to keep it linear

What does migration distance tell us?

How far the proteins migrated from their initial position (well of the gel) towards the anode

What happens with heavier/bigger proteins? (migration distance and migration rate)

harder for proteins to pass through the pores = shorter migration distance = slower migration rate

What is the migration rate?

how fast the molecules travel to the anode

How do we estimate the size of the protein?

Look at the protein marker/ladder

What is the measure of molecular weight/mass

Dalton

Dalton is equivalent to?

The mass of 1 hydrogen atom in g/mol

How do you contrast a protein marker from a DNA marker?

Bands of DNA markers do not have identities, just known sizes

What are the types of SDS-PAGE? Describe.

1. Continuous → one type of gel

2. Discontinuous → stacking gel on top, resolving/running gel below

12 components of SDS-PAGE

1. Buffer tank/chamber

2. Electrode assembly

3. Tank lid with power cables

4. Power supply

5. Casting stand

6. Casting frame

7. Gasket

8. Gel releasers

9. Sample loading guide

10. Spacer plates

11. Glass plates

12. Comb

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Ideal stacking gel concentration

4%

6 components of the stacking gel

1. Monomer solution

2. Stacking gel buffer

3. Deionized, distilled water

4. 10% SDS

5. 10% APS

6. TEMED

“Most Gel Buffers Drop 10% Smoothly Amid Turbulence”

What does the stacking gel do?

It’s the upper portion of a discontinuous polyacrylamide gel that concentrates or stacks the components of the sample to create a very thin starting zone or band

What is the ideal concentration of the resolving gel?

5-15%

Which of the two gels has a higher concentration/smaller pores?

Resolving/running gel has smaller pores and higher gel concentration

Monomer solution has two components:

1. Acrylamide

2. Bis-acrylamide

What is the percentage and ratio of the components of the monomer solution

30% acrylamide/bis-acrylamide solution, 29:1

What is acrylamide for?

a monomer used with a cross-linker to form the matrix used for separating proteins

What is bis-acrylamide for?

a common cross-linker used with acrylamide to form a support matrix

What is the concentration of the running gel buffer and what is it for?

(1.5 M Tris-HCI, pH 8.8)

• provide an environment near physiological and maintain pH

What is the concentration of the stacking gel buffer and what is it for?

(0.5 M Tris-HCI, pH 6.8)

• It is ideal to stack proteins by having a lesser concentration of this

What is constant weight ratio that SDS binds to polypeptides in?

1:4 (SDS:polypeptide)

What does APS stand for?

ammonium persulfate

What does TEMED stand for?

Tetramethylethylenediamine

Function of APS and TEMED

Radical initiators to catalyze the polymerization through cross-linking of acrylamide and bis-acrylamide in making a polyacrylamide gel

5 components of the sample/reducing treatment buffer

1. Deionized H2O

2. Tris-HCl pH (6.8–7.0)

3. 10% SDS

4. β-mercaptoethanol

5. 1% bromophenol blue

“Daring Tigers Stand Boldly and Bravely“

Function of deionized H2O

Primary vehicle/solvent

Function of Tric-HCl with pH 6.8—7.0

Maintaints optimum pH of the solution and prevents protein degradation

Function of SDS in sample buffer

Makes the net charge of the protein negative

Function of 2-mercaptoethanol in sample buffer

A reducing agent that cleaves disulfide bonds to achieve complete protein unfolding and to maintain proteins in a fully reduced state

Function of 1% bromophenol blue

To track the movement of proteins towards the positive electrode

Function of glycerol

Denser than water to keep samples from floating out of the well

Function of running (Tris/HCl/glycine/SDS) or tank buffer

Provides the ions for the electrical current in an electrophoresis runs

Function of Tris-HCl, glycine, SDS in running/tank buffer

1. SDS → denaturing agent

2. Glycine → provides the trailing ions so glycine in the stacking gel can stack or compress the proteins with the chloride ion

3. Tris-HCl → maintain optimum conditions

3 components of R-250 CBB

Methanol, Glacial Acetic Acid, and H2O

What is destaining the polyacrylamide gel for?

Removes excess stain for a higher resolution of the polyacrylamide image

What are the stacking gel components and their quantities?

___ the polyacrylamide gel removes excess stain for a higher resolution of the polyacrylamide image

De-staining

3 components in G-250 variant?

1. Methanol

2. Glacial Acetic Acid

3. H₂O