Key Difference – SDS Page vs Western Blot
Western blotting is a technique which detects a specific protein from a protein sample. This technique is performed via several key steps: gel electrophoresis, blotting, and hybridization. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS Page) is a kind of gel electrophoresis technique which is used to separate proteins according to their sizes (molecular weights). Western blot is a special sheet of a blotting membrane which is used to transfer the same pattern of the proteins in the SDS Page. The key difference between SDS Page and western blot is that SDS Page allows the separation of proteins in a mixture while western blot allows detection and quantification of a specific protein from a mixture. Both are useful in protein analysis studies.
CONTENTS
1. Overview and Key Difference
2. What is SDS Page
3. What is Western Blot
4. Side by Side Comparison – SDS Page vs Western Blot
5. Summary
What is SDS Page?
SDS Page is a gel electrophoresis technique used for protein separation. It is commonly used in biochemistry, genetics, forensics and molecular biology. Once the proteins are extracted from the sample, they are run on a gel made up of SDS and polyacrylamide. SDS is an anionic detergent which is used to linearize proteins (denature proteins) and to impart a negative charge to linearized proteins proportional to their molecular mass. Polyacrylamide becomes the solid support for the gel. Denatured proteins which are negatively charged migrate towards the positive end of the apparatus through the gel. According to the sizes of the proteins, migrating speeds differ among proteins and separation occurs. Therefore, SDS page is useful for the separation of individual proteins based on their sizes.
The preparation of the polyacrylamide gel for the fractionation of proteins is a crucial step in SDS Page. The correct concentration of polyacrylamide and the type of the cross-linking agent used strongly affect the physical properties of the gel, which makes the actual separation of the different proteins. The pore sizes of the gel should be properly managed for effective separation. However, SDS Page is considered as a high-resolution protein separation technique.
SDS Page technique has a major limitation in protein analysis. Since SDS denatures proteins prior to separation, it does not allow the detection of enzymatic activity, protein binding interactions, protein cofactors, etc.
Figure 01: SDS Page
What is Western Blot?
Western blotting technique enables the detection of a specific protein from a protein mixture and measurement of the quantity and molecular weight of the protein. Western blot is the membrane used during the blotting procedure to get the mirror image of the protein patterns in the SDS- polyacrylamide gel. The membrane which is used for western blotting is mostly made up of nitrocellulose or polyvinylidene difluoride (PVDF). The membrane with the transferred protein can be used to identify the desired protein. It requires high-quality antibody for the detection of the desired protein by hybridization. The antibody binds to its specific antigen and reveals the presence of the desired antigen which is a protein.
Transferring of the proteins from the SDS polyacrylamide gel to the western blot is performed by electroblotting. It is an effective and fast method which causes the proteins to electrophorese out of the gel and pass onto the nitrocellulose membrane (western blot).
Figure 02: Western Blot
What is the difference between SDS Page and Western Blot?
SDS Page vs Western Blot |
|
| SDS Page is a gel electrophoresis technique. | Western blot is a technique which is performed on a membrane to detect a specific protein from a mixture. |
| Use | |
| SDS page allows separation of proteins according to their sizes. | Western blot allows the transferring of proteins on the SDS page gel without changing its pattern & allows hybridization with specific antibodies. |
| Disadvantages | |
| Protein denaturation, high-cost, and presence of neurotoxin chemicals are the disadvantages of this technique. | This technique is time-consuming and requires a good experienced personal and specific controlled conditions. |
Summary – SDS Page vs Western Blot
SDS page and western blot are two methods involved in protein analysis. SDS Page allows easy separation of proteins on a gel according to their molecular weight. Western blot helps to confirm the presence and quantity of a specific protein through hybridization with specific antibodies. This is the difference between SDS Page and western blot.
References:
1. Blancher, C., and A. Jones. “SDS -PAGE and Western Blotting Techniques.” Methods in molecular medicine. U.S. National Library of Medicine, n.d. Web. 27 Mar. 2017
2. Nowakowski, Andrew B., William J. Wobig, and David H. Petering. “Native SDS-PAGE: High Resolution Electrophoretic Separation of Proteins With Retention of Native Properties Including Bound Metal Ions.” Metallomics: integrated biometal science. U.S. National Library of Medicine, May 2014. Web. 27 Mar. 2017.
Image Courtesy:
1. “SDS-PAGE Electrophoresis” By Bensaccount at English Wikipedia (CC BY 3.0) via Commons Wikimedia
2. “Western blot 114A” By Amanthabagdon – Own work (Public Domain) via Commons Wikimedia