Table of Contents
Before we dive into the details, let's take a look at the structure of today's presentation.
We will begin with an Introduction that covers the importance and applications of SDS-PAGE in protein
analysis. Then, we’ll move into the 1D technique, where I’ll explain the principles of 1D SDS-PAGE,
followed by real-world examples in the 1D example section.
Next, we will transition to the 2D technique, where I will explain the principles and processes of 2D SDS-
PAGE, accompanied by scientific examples in the 2D example section.
In the Comparison section, I’ll compare the strengths and weaknesses of 1D and 2D SDS-PAGE, and
finally, I’ll conclude with some Tips and Tricks to help ensure the effective use of SDS-PAGE.
Slide 1: Introduction to Protein Separation Methods
Good afternoon,
My name is Phan Nam Hải, and today, our group will be discussing protein separation methods, with a
focus on SDS-PAGE. SDS-PAGE, or Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, is a
powerful tool used in the separation and analysis of proteins. Through today’s presentation, we will
explore both 1D and 2D SDS-PAGE techniques, understanding their principles, applications, and how
they contribute to scientific research.
Slide 2: Overview of 1D SDS-PAGE
In 1D SDS-PAGE, the main objective is to separate proteins based solely on their molecular weight. The
technique begins with treating proteins using SDS, a detergent that denatures proteins by unfolding
them and coating them with a uniform negative charge.
Once the proteins are denatured, an electric field is applied across a polyacrylamide gel, which acts as a
molecular sieve. Smaller proteins migrate faster through the gel, while larger proteins move more slowly,
resulting in the separation of proteins based on size. This process is widely used in protein purity checks
and protein expression analysis, making it invaluable in research and clinical applications.
Slide 3: Protein Separation Process in 1D SDS-PAGE
This is an illustrative image of the 1D SDS-PAGE process. The arrow indicates the direction of protein
migration, showing that proteins move from the negative cathode to the positive anode under the
influence of an electric field.
Slide 4: Overview of 2D SDS-PAGE
Unlike 1D SDS-PAGE, which separates proteins only by size, 2D SDS-PAGE introduces a second dimension
of separation: the isoelectric point (pI).
This is an illustrative image of a 2D SDS-PAGE gel. The two arrows here represent the molecular weight
(MW) and isoelectric point (pI) of the circled protein spot. In this example, the protein has a molecular
weight of approximately 70 kDa and a pI of 6, indicating its position on the gel based on these two
properties.
In the first step, proteins are separated by their pI using Isoelectric Focusing (IEF). This allows proteins
with similar molecular weights but different pI values to be distinguished. The second step involves
applying SDS-PAGE, where proteins are further separated based on their size. This two-step process
provides greater resolution and enables the detection of subtle differences between proteins, making 2D
SDS-PAGE especially useful in proteomics."
Slide 5: This slide shows how SDS and DTT denature proteins and break disulfide bonds, ensuring
complete unfolding for accurate size-based separation.
Slide 6: Here, we see the role of Tris-buffer, acrylamide, and IPG strips in stabilizing pH, forming the gel
matrix, and aiding 2D separation by pI.
Slide 7: In summary, these reagents are critical for ensuring precise protein separation in both 1D and 2D
SDS-PAGE.
