How to Choose the Optimal Gel Concentration and Buffer pH for Protein SDS-PAGE and Molecular Weight Determination
Gel Concentration Selection
1. The choice of gel concentration should be based on the molecular weight range of the protein to be tested. Generally, lower gel concentrations are suitable for larger proteins, while higher concentrations are better for smaller proteins.
2. Commonly used gel concentrations range from 8% to 15%. For larger proteins, a lower concentration such as 8% is recommended, while for smaller proteins, higher concentrations like 12% or 15% are ideal.
3. If the molecular weight of the protein is unknown, different gel concentrations can be tested to determine the optimal one.
Buffer pH Selection
1. The pH of the buffer plays an important role in the electrophoretic mobility and separation efficiency of proteins. A suitable buffer pH can ensure good separation of proteins in the gel.
2. A common buffer system is the Tris-Glycine buffer, with a pH range of 8.3-8.8. This range is suitable for most proteins.
3. If the protein is prone to degradation or aggregation under alkaline conditions, a lower buffer pH, such as 8.3, is preferred. If the protein is prone to degradation or aggregation under acidic conditions, a higher buffer pH, such as 8.8, is preferred.
4. If the optimal buffer pH is uncertain, different pH values can be tested to determine the best one.
Choosing the optimal gel concentration and buffer pH requires consideration of the protein's molecular weight range, sample characteristics, experimental goals, and the stability and reproducibility of the experimental conditions. By testing different gel concentrations and buffer pH values, the best conditions can be determined.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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