Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a commonly used technique for protein purification analysis. SDS-PAGE separates proteins based on differences in charge and molecular size, resulting in different migration rates. If a protein sample contains multiple proteins or if a purified protein sample contains other contaminant proteins, different proteins will be separated into multiple protein bands after SDS-PAGE separation. If the purified sample contains only one type of protein, only one protein band will appear after electrophoresis. Therefore, SDS-PAGE can be used to analyze the purity of protein samples.
Principle of SDS-PAGE analysis
SDS is an anionic surfactant that can break hydrogen and hydrophobic bonds in proteins and binds with protein molecules in a fixed ratio to form complexes. This causes the proteins to carry a negative charge far exceeding their original charge, masking the natural charge differences between various protein molecules. As a result, the migration speed of protein-SDS complexes during electrophoresis is determined only by the protein's molecular weight. Different proteins are separated based on molecular weight differences after SDS-PAGE electrophoresis, and the separation results are analyzed by protein staining.
Protein SDS-PAGE analysis process
1. Protein concentration detection 2. Sample preparation: Add an equal amount of 2x loading buffer containing β-mercaptoethanol to the protein sample and boil for 10 minutes
3. Electrophoresis preparation: Prepare an SDS separating gel of appropriate concentration, set up the electrophoresis tank, and pour in 1x electrophoresis buffer
4. Load protein samples:Based on protein concentration, add an appropriate amount of treated protein sample sequentially into the electrophoresis wells, and add an appropriate amount of protein standard markers into other empty wells.
5. Start electrophoresis: Connect the power supply and set the voltage to 120V, maintaining a constant voltage.
6. Gel removal after electrophoresis: Stop electrophoresis when the bromophenol blue reaches the bottom of the gel; remove the protein gel and cut off the stacking gel and bromophenol blue strip at the bottom of the separating gel.
7. Staining: Stain the protein gel with R250 staining solution for 1 hour
8. Destaining: Use destaining solution to destain the stained protein gel until the background is clean and the protein bands are clearly visible
9. Photograph and analyze
Bilingual project report
In the technical report, Biotye Parker will provide you with a detailed bilingual technical report in both Chinese and English, including:
1. Experimental steps (in Chinese and English)
2. Relevant SDS-PAGE parameters (in Chinese and English)
3. Protein purity results
One-stop service for SDS-PAGE protein purity analysis
You just need to place an order and send samples
Biotye Parker's one-stop service includes: sample preparation - on-machine analysis - data analysis - project report
