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1. Introduction to Biochemistry4h 34m
2. Water3h 23m
3. Amino Acids8h 10m
4. Protein Structure10h 4m
5. Protein Techniques14h 5m
6. Enzymes and Enzyme Kinetics13h 38m
7. Enzyme Inhibition and Regulation 8h 42m
8. Protein Function 9h 41m
9. Carbohydrates7h 49m
10. Lipids5h 49m
11. Biological Membranes and Transport 6h 37m
12. Biosignaling9h 45m
Review 1: Nucleic Acids, Lipids, & Membranes2h 47m
Review 2: Biosignaling, Glycolysis, Gluconeogenesis, & PP-Pathway3h 12m
Review 3: Pyruvate & Fatty Acid Oxidation, Citric Acid Cycle, & Glycogen Metabolism2h 26m
Review 4: Amino Acid Oxidation, Oxidative Phosphorylation, & Photophosphorylation1h 48m

5. Protein Techniques

Native Gel Electrophoresis

5. Protein Techniques

Native Gel Electrophoresis: Videos & Practice Problems

Video Lessons Practice Worksheet

Topic summary

Native gel electrophoresis, or native PAGE, separates proteins based on their native charges, shapes, and sizes using an electric field. Proteins migrate towards opposite charges, with larger proteins moving slower. The migration is influenced by mass, charge, and shape, allowing for the retention of native properties. After staining, proteins appear as bands, with thickness indicating quantity. Understanding these factors is crucial for analyzing protein behavior, as differences in charge and shape can lead to varied migration patterns, making native PAGE a valuable tool in proteomics.

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Native Gel Electrophoresis

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Native Gel Electrophoresis Video Summary

Native gel electrophoresis, also known as native polyacrylamide gel electrophoresis (native PAGE), is a technique used to separate proteins based on their native charges, shapes, and sizes. This method utilizes an electric field to facilitate the migration of charged proteins through a gel matrix made of polyacrylamide. The electric field is generated by connecting a power supply to the gel, creating a negative charge at one end and a positive charge at the other. Proteins with a native charge will migrate towards the opposite charge; negatively charged proteins move towards the positive electrode, while positively charged proteins move towards the negative electrode.

In native PAGE, the migration of proteins is influenced by three key factors: charge, size, and shape. Larger proteins tend to migrate more slowly through the gel compared to smaller proteins, which can travel further in the same amount of time. Additionally, the native shape of the proteins is preserved during this process, which can significantly affect their migration patterns. For instance, two proteins with identical mass and charge may migrate differently if their shapes differ. This highlights the importance of considering all three factors when analyzing protein migration in native PAGE.

After running the gel, proteins are visualized as distinct bands, with the intensity of each band indicating the quantity of the corresponding protein. A thicker band suggests a higher concentration of that protein, while a thinner band indicates a lower concentration. For example, if proteins A, B, and C are analyzed, even if they have the same mass, their differing charges and shapes will result in varied migration distances, leading to distinct banding patterns on the gel.

Ultimately, native PAGE is beneficial for studying proteins in their natural state, as it allows for the retention of their native properties. However, the complexity introduced by the interplay of charge, size, and shape can make it challenging to predict migration outcomes. This complexity is one reason why other methods, such as SDS-PAGE, are often employed, as they simplify the analysis by focusing on a single factor influencing migration.

Problem

Which advantage does native gel electrophoresis provide as a protein technique?

Allows separation of all native proteins as they migrate through the gel.

Allows separation of all native protein subunits based on their size (large proteins travel slower).

Native proteins always migrate through the electric field towards the positive end.

Separates charged proteins while allowing them to retain their native conformation.

Problem

Which option below best describes the native gel electrophoresis migration for Proteins A, B, C & D (assuming equal mass & shape) considering that the buffer solution has a pH = 6.4.
Protein A pI = 5.2, Protein B pI = 6.4, Protein C pI = 7.0, Protein D pI = 9.2

A & B will migrate to the negative pole while C & D migrate to the positive pole.

A will migrate to the positive pole, B will not migrate, while C & D migrate to the negative pole.

A & B will migrate to the positive pole while C & D migrate to the negative pole.

A will migrate to the negative pole, B will not migrate, while C & D migrate to the positive pole.

Problem

A) Consider both the peptide Gly—Pro—Ser—Glu—Thr (in a linear chain) and a cyclic peptide of the same exact sequence Gly—Pro—Ser—Glu—Thr (with a peptide bond linking the Thr & Gly). Are these peptides chemically the same? Explain.

B) Can you expect to separate the peptides above by Native-PAGE? Why or why not?

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Native Gel Electrophoresis Practice 3 Video Summary

In this practice problem, we explore the differences between a linear peptide and a cyclic peptide composed of the amino acids glycine, proline, serine, glutamate, and threonine. The linear peptide maintains a straightforward sequence, while the cyclic peptide features an additional peptide bond that connects the terminal threonine to the initial glycine, forming a loop. This structural distinction leads to significant chemical differences between the two peptides.

To understand whether these peptides are chemically the same, we must consider the formation of peptide bonds, which occurs through dehydration synthesis. This process involves the reaction of the carboxylate group of threonine with the amino group of glycine, resulting in the release of a water molecule (H₂O). Consequently, the cyclic peptide is missing this water molecule, indicating that it has fewer atoms compared to the linear peptide. Additionally, the cyclic structure lacks free or ionizable amino and carboxyl groups, which are present in the linear peptide. Therefore, the cyclic peptide is not chemically identical to the linear peptide due to these differences in composition and functional groups.

Moving on to the second part of the problem, we consider whether these peptides can be separated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE separates proteins based on their native properties, including shape, mass, and charge. The cyclic and linear peptides differ significantly in shape due to the cyclic structure of the former, which allows for their separation. Furthermore, the cyclic peptide has a lower mass because it lacks the water molecule present in the linear peptide. This difference in mass contributes to their separation during electrophoresis.

Additionally, the linear peptide possesses more ionizable groups, which can affect its charge depending on the pH of the environment. This variability in charge further distinguishes the two peptides, enhancing the likelihood of their separation in a native PAGE setup. In summary, the differences in shape, mass, and potential charge variations due to pH make it feasible to separate these peptides using native PAGE.

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What is native gel electrophoresis and how does it work?

Native gel electrophoresis, also known as native PAGE, is a technique used to separate proteins based on their native charges, shapes, and sizes using an electric field. In this method, proteins retain their native structures and charges, which influence their migration through the gel. The gel matrix is typically made of polyacrylamide. Proteins with a native charge will migrate towards the opposite charge; negatively charged proteins move towards the positive electrode, and positively charged proteins move towards the negative electrode. Larger proteins move slower through the gel, while smaller proteins move faster. After staining, proteins appear as bands, with the thickness of the bands indicating the quantity of the proteins.

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What factors influence protein migration in native gel electrophoresis?

In native gel electrophoresis, protein migration is influenced by three main factors: mass, charge, and shape. Larger proteins migrate more slowly through the gel, while smaller proteins move faster. The native charge of the protein determines its direction of movement; negatively charged proteins move towards the positive electrode, and positively charged proteins move towards the negative electrode. Additionally, the shape of the protein affects its migration, as proteins with different shapes but the same mass and charge can migrate differently through the gel. These factors combined make native PAGE a complex but valuable tool for analyzing protein behavior.

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How do you interpret the bands on a native PAGE gel?

After running a native PAGE gel and staining it, proteins appear as bands. The position of the bands indicates the relative migration of the proteins, influenced by their mass, charge, and shape. Larger proteins will be higher in the gel, while smaller proteins will be lower. The thickness of the bands indicates the quantity of the proteins; thicker bands represent a higher quantity, while thinner bands represent a lower quantity. By comparing the bands, you can infer differences in protein properties and quantities within your sample.

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What are the advantages of using native gel electrophoresis?

Native gel electrophoresis offers several advantages. It allows proteins to retain their native structures, charges, and interactions, providing a more accurate representation of their natural state. This is particularly useful for studying protein complexes and interactions. Additionally, native PAGE can separate proteins based on multiple factors (mass, charge, and shape), offering a comprehensive analysis of protein properties. However, this complexity can also make it more challenging to predict migration patterns compared to other methods like SDS-PAGE, which focuses solely on mass.

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How does native gel electrophoresis differ from SDS-PAGE?

Native gel electrophoresis (native PAGE) and SDS-PAGE are both techniques used to separate proteins, but they differ significantly. Native PAGE separates proteins based on their native charges, shapes, and sizes, allowing them to retain their native structures and interactions. In contrast, SDS-PAGE denatures proteins and coats them with a uniform negative charge, separating them solely based on their mass. This makes SDS-PAGE simpler to interpret, as only one factor (mass) influences migration. Native PAGE, however, provides a more comprehensive analysis of protein properties, making it valuable for studying native protein behavior and interactions.

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