Medical Biochemistry I - Lab Exercise 3: SDS-PAGE

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Questions and Answers

What is the primary purpose of SDS-PAGE in molecular biology?

To increase the pH of protein samples.

To enhance protein solubility in solutions.

To preserve the tertiary structure of proteins.

To separate proteins based on their molecular weight. (correct)

Which component of the Laemmli Sample Buffer is responsible for protein denaturation?

Bromophenol Blue

Tris-HCl

SDS (correct)

Glycerol

In SDS-PAGE, how do proteins migrate in the gel when an electric field is applied?

Toward the negatively charged electrode.

From anode to cathode regardless of size.

From cathode to anode, with low molecular weight proteins migrating faster. (correct)

Based on their charge, regardless of molecular weight.

What role do reducing agents play in the SDS-PAGE process?

They cleave disulfide bonds to help denature proteins. Signup and view all the answers

What type of gel is typically used in SDS-PAGE?

Discontinuous polyacrylamide gel. Signup and view all the answers

During the staining process in SDS-PAGE, what is the purpose of the staining buffer?

To bind and visualize the proteins in the gel. Signup and view all the answers

Which of the following statements about SDS-PAGE is TRUE?

Proteins are coated with SDS to ensure uniform negative charge. Signup and view all the answers

Which of the following proteins would migrate furthest during an SDS-PAGE analysis?

A 10 kDa protein. Signup and view all the answers

What is the primary reason for heating protein samples immediately after the addition of Laemmli buffer?

To prevent degradation of denatured proteins by proteases. Signup and view all the answers

How much loading buffer should be added relative to the total sample volume?

One-fourth of the sample volume. Signup and view all the answers

Why is it essential to connect the anode to the bottom chamber during electrophoresis?

To allow the negatively charged proteins to migrate towards the anode. Signup and view all the answers

What is the optimal voltage setting for running the gels?

180 volts, adjusted empirically. Signup and view all the answers

What is the purpose of adding Coomassie Blue staining solution after running the gel?

To visualize the proteins within the gel. Signup and view all the answers

What is the consequence of overheating the gel during electrophoresis?

Distortion of acrylamide and potential cracking of plates. Signup and view all the answers

What should be done immediately after the dye front reaches the bottom of the gel?

Stop the run and turn off the power. Signup and view all the answers

How often should the Destain solution be changed during the incubation period?

Twice during the 15 minutes. Signup and view all the answers

Study Notes

MD100 Medical Biochemistry I - Lab Exercise 3: Introduction to SDS PAGE

Course: Medical Biochemistry I
Lab Exercise: Introduction to SDS PAGE, Protein identification and characterization
Semester: Fall 2024
Institution: European University Cyprus, School of Medicine

Objectives

SDS-PAGE: Introduction to the laboratory technique, theoretical background (principle of SDS-PAGE).
Parts: Breakdown of the lab exercise into parts: sample preparation (including dilutions), sample loading and running gel, gel staining and destaining, protein gel analysis (protein identification).

Introduction to SDS-PAGE

Technique: Used to separate proteins based on their molecular weight through electrophoresis.
Electrophoresis: The separation of macromolecules in an electric field is called electrophoresis.
SDS-PAGE: In SDS-PAGE, a polyacrylamide gel is the support medium and sodium dodecyl sulfate (SDS) is used to denature the proteins.
Principle: A charged molecule migrates to the oppositely charged electrode when placed in an electric field. SDS, working as an anionic detergent, denatures and binds to proteins, uniformly giving them a negative charge. This ensures all SDS-bound proteins migrate in the same direction (toward the anode) when current is applied, regardless of their initial charge.
SDS effect: Reducing agents (e.g., β-mercaptoethanol) cleave disulphide bonds and disrupt protein structure.

The principle of SDS-PAGE (detailed)

Protein structure: Proteins initially have complex folded structures with positive and negative charges.
Reduction: Reducing agents like 2-mercaptoethanol (2-ME) break the disulfide bonds in proteins and cause them to unwind.
SDS binding: SDS molecules bind to the unfolded proteins and impart a uniform negative charge, which is proportional to the length of the polypeptide chain. This effectively negates any initial positive and negative charges of the proteins.
Migration: When the protein mixture is subjected to an electric field, the negatively charged protein-SDS complexes will migrate towards the anode (positive electrode), with smaller proteins travelling faster than larger ones due to less obstruction within the polyacrylamide gel matrix.
Molecular weight separation: This allows separation of proteins based on their molecular weight—smaller proteins migrate a greater distance along the gel.

Protein Identification

Albumin: Molecular weight: 66.5 kDa.
Casein: Molecular weight: 24 kDa.

Materials/Equipment

Gels: Prepared in lab or pre-cast.
Electrophoresis Chamber: Vertical gel electrophoresis chamber.
Protein Samples: Sample solutions for analysis.
Buffers: Running buffer (Tris/Glycine/SDS), staining/destaining buffers.
Protein Ladder: Pre-stained protein molecular weight standards.
Micropipettes/tips: For accurate sample/buffer delivery.
Laemmli Sample Buffer: Used for sample preparation, containing components like Tris-HCl buffer, SDS, glycerol, 2-mercaptoethanol, and bromophenol blue.

Sample Preparation

Dilutions: Create 5%, 2.5%, and 1% dilutions of 10% unknown protein sample.
Laemmli Buffer Addition: Add 5 µL of 4x Laemmli stock buffer to each dilution. The important note here is that the Laemmli sample buffer should be ¼ of the sample volume.
Heating for Protein Denaturation: Heat the samples at 70°C in a heat block for 2 min to disrupt the protein structure. SDS enhances this denaturing function and renders the resulting proteins uniformly negatively charged

Protocol

Dilutions: Prepare dilutions of unknown protein solution with water.
Sample Addition: Add precise volumes of each dilution to new labeled tubes.
Laemmli Addition: Add Laemmli Sample Buffer to each tube.
Heat Denaturation: Boil the mixed samples to denature proteins in a heat block at 70°C for 2 min.
Buffer Addition: Add running buffer to both chambers of the electrophoresis apparatus.
Ladder Loading: Load the protein ladder in the first lane of the gel.
Sample Loading: Load other protein samples in the following lanes.
Electrophoresis: Run the gel at 180 volts for roughly 30 min, until the dye front reaches the bottom of the gel.
Gel Staining: Remove the separated proteins from the gel and transfer it to a staining dish filled with deionized water and Coomassie Blue staining solution for 15 minutes with rocking.
Destaining: Remove the Coomassie blue staining solution, rinse with deionized water multiple times.
Overnight Destain:* Remove the staining solution and fill the staining dish with deionized water, incubating the gel overnight while rocking to ensure complete removal.
Destain Changes: Perform two washes, each about 15 minutes, for total 30 minutes of destaining. Change the destaining solution with fresh solution every 5 mins.
Gel Picture: Take a picture of the gel to analyse.

Protein gel analysis

Band interpretation: Bands in the gel indicate the presence of proteins or protein subunits. Single bands for a lane suggest one protein/protein subunit; multiple bands mean multiple proteins or subunits/polypeptides.
Protein identification: Determine protein identity by comparing the migration distances of unknown sample bands to the bands from known proteins (like the protein ladder).
Protein concentration: Assess protein concentration by the intensity (darkness) of the bands.

Additional

Question 1: The correct answer is C. Smaller proteins migrate faster due to less obstruction through the gel than larger ones
Question 2: The correct answer is A. Proteins are visualized using dye staining on gels
Results: Sample gel image illustrating the identified protein bands.
Protein Ladder: The protein ladder is used to gauge the molecular weight of unknown proteins in the samples.

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Description

This quiz covers Lab Exercise 3 of Medical Biochemistry I, focusing on the introduction to SDS-PAGE. Learn about the theoretical background, sample preparation, gel loading, and analysis of protein identification through this essential electrophoresis technique.