SDS-PAGE Protein Purity Analysis: How to Analyze Purity?
SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a commonly used method for protein analysis. By comparing the relative migration distance of proteins, it can be used to analyze the size of proteins. By observing the number and intensity of bands on the gel, protein purity can be roughly assessed. The basic steps for evaluating protein purity using SDS-PAGE are as follows:
1. Preparation of polyacrylamide gel:
Prepare a polyacrylamide gel of appropriate concentration, usually selected based on the size of the protein. For most applications, a concentration of 8-15% is suitable for most proteins. For higher resolution applications, gradient gels can be used.
2. Sample loading:
Load the treated protein samples into the wells of the gel. A molecular weight standard (molecular weight marker) should also be loaded for subsequent analysis of protein size.
3. Electrophoresis:
Conduct electrophoresis at a certain voltage to allow proteins to migrate along the electric field direction in the gel. The electrophoresis time depends on the gel concentration, voltage, and protein size.
4. Staining and destaining:
After electrophoresis, stain the gel, commonly using Coomassie Brilliant Blue or silver staining methods. Then perform destaining to remove excess dye, allowing for clear observation of protein bands.
5. Result analysis:
Observe the protein bands on the gel and assess protein purity. Ideally, a pure protein should appear as a single distinct band. If multiple bands are present, it indicates impurities, and image analysis software can be used to quantitatively analyze the intensity of the bands to evaluate the purity of the target protein.
It should be noted that purity determination by SDS-PAGE is a qualitative or semi-quantitative method used for quickly assessing the purity of protein samples. For more precise purity evaluation, methods such as high-performance liquid chromatography (HPLC) can be considered.
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