Key Difference – Stacking Gel vs Separating Gel
The terms stacking gel and separating gel are used in explaining the SDS-PAGE technique. SDS-PAGE or sodium dodecyl sulfate-polyacrylamide gel electrophoresis is a laboratory technique that is used to separate protein molecules based on their molecular weights. The theory behind the technique is that proteins with different molecular weights show different migration rates; low molecular weight molecules migrate faster whereas high molecular weight molecules migrate slowly. The medium for migration is a gel. There are two types of gels named as stacking gel and separating gel. The key difference between stacking gel and separating gel is that the pH of the stacking gel is 6.8 whereas the pH of the separating gel is 8.8.
CONTENTS
1. Overview and Key Difference
2. What is Stacking Gel
3. What is Separating Gel
4. Relationship Between Stacking Gel and Separating Gel
5. Side by Side Comparison – Stacking Gel vs Separating Gel in Tabular Form
6. Summary
What is Stacking Gel?
Stacking gel is a low concentrated polyacrylamide gel that is placed on the top of more concentrated resolving gel (separating gel) in SDS-PAGE technique. The stacking gel is used to improve the resolution of electrophoresis. The resolution increases because of the difference between concentrations of stacking gel and resolving gel that effect on the proteins in the sample. Since the concentration of polyacrylamide in stacking gel is low, the pore size is higher. This also helps in increasing the separation.
The stacking gel is a loose polyacrylamide gel with big pores of about 7% polyacrylamide. These pores do not act as a considerable barrier for large protein molecules. Hence this gel affects slightly on the mobility of those proteins. This makes separation according to the mobility and size of protein by concentrating them in between two gels.
Figure 01: SDS-PAGE Acrylamide Gel
The pH of the stacking gel is 6.8. Its pH is acidic than that of resolving gel by 2 pH units. This pH implies a lower ionic strength hence a higher electrical resistance. This provokes the mobility of proteins than other charged particles present in the gel.
What is Separating Gel?
Separating gel or resolving gel of an SDS-PAGEtechnique is a highly concentrated polyacrylamide gel that is placed on the top of low concentrated stacking gel. The separating gel contains a high amount of polyacrylamide (10%). The pH of this gel is maintained as pH=8.8, which is a higher pH level than that of the stacking gel.
Figure 02: A Gel Electrophoresis Apparatus
When the protein molecules reach the separating gel, the migration of those molecules is slowed down because the separating gel is a high concentration gel with a small pore size that can act as a considerable barrier for the movement of the protein molecules. This slow down allows the other proteins are migrating slowly to catch up, resulting in a narrow, concentrated band in between the two gels.
What is the Relationship Between Stacking Gel and Separating Gel?
- Both Stacking Gel and Separating Gel types are used in the same apparatus.
What is the Difference Between Stacking Gel and Separating Gel?
Stacking Gel vs Separating Gel |
|
| Stacking gel is a low concentrated polyacrylamide gel that is placed on the top of more concentrated resolving gel (separating gel) in SDS-PAGE technique. | Separating gel or resolving gel of an SDS-PAGE technique is a highly concentrated polyacrylamide gel that is placed on the top of a low concentrated stacking gel. |
| Placement | |
| Stacking gel is placed on the resolving (separating) gel. | Separating gel is placed on the bottom of the container used for gel electrophoresis. |
| Concentration | |
| The concentration of stacking gel is high. | The concentration of separating gel is low. |
| Polyacrylamide Content | |
| Stacking gel contains about 7% polyacrylamide. | Separating gel contains about 10% polyacrylamide. |
| pH | |
| The pH of the stacking gel is 6.8. | The pH of the separating gel is 8.8. |
| Pore Size | |
| Large pore sizes are present in stacking gel. | Small pore sizes are present in separating gel. |
| Resolution | |
| Stacking gel gives better resolution. | Separating gel gives a poor resolution. |
Summary – Stacking Gel vs Separating Gel
Stacking gel and separating gel are two types of polyacrylamide gels used to get better separation of protein molecules in a given sample. The difference between stacking gel and separating gel is that the pH of the stacking gel is 6.8 whereas the pH of the separating gel is 8.8.
Reference:
1.“ Polyacrylamide Gel Electrophoresis.” Polyacrylamide Gel Electrophoresis (Theory) : Molecular Biology Virtual Lab II : Biotechnology and Biomedical Engineering : Amrita Vishwa Vidyapeetham Virtual Lab. Available here
2.“How SDS-PAGE Works.” Bitesize Bio, 16 Feb. 2018. Available here
3.“SDS-PAGE stacking gel – BICH 608 (Polymenis-Pettigrew).” Google Sites. Available here
Image Courtesy:
1.’SDS-PAGE Acrylamide gel’By Bensaccount at English Wikipedia, (CC BY 3.0) via Commons Wikimedia
2.’Gel electrophoresis apparatus’ By Jeffrey M. Vinocur – Own work, (CC-BY 2.5) via Commons Wikimedia
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