Molecular Weight and Isoelectric Point Analysis of Proteins

Molecular Weight Analysis

1. SDS-PAGE (Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis)

SDS-PAGE is a commonly used method for determining protein molecular weight. By conducting electrophoresis in a gel containing SDS (sodium dodecyl sulfate), proteins separate according to their molecular weight. After electrophoresis, proteins can be visualized through staining, and by comparison with standard proteins of known molecular weight, the molecular weight of sample proteins can be estimated.

 

2. Mass Spectrometry (MS)

Mass spectrometry is a high-precision method for determining protein molecular weight. By measuring the mass-to-charge ratio (m/z) of proteins or protein fragments, the molecular weight of proteins can be determined with high accuracy. Mass spectrometry can also be used for protein identification and characterization.

 

Isoelectric Point (pI) Analysis

1. Isoelectric Focusing (IEF)

Isoelectric focusing is a separation technique based on the isoelectric point of proteins. In this method, proteins migrate in a pH gradient until they reach their isoelectric point (pI), the pH at which the protein no longer carries a charge. At this point, protein migration stops. By comparing with standards of known pI values, the isoelectric point of the sample protein can be determined.

 

2. 2D Electrophoresis

2D electrophoresis first separates protein samples by isoelectric focusing, and then further separates them by molecular weight in the second dimension using SDS-PAGE. This method can provide both the isoelectric point and molecular weight information of proteins, making it very suitable for proteomic analysis of complex samples.

 

Data Analysis Software

For the computation of protein molecular weight and isoelectric point, various proteomics databases and software tools can be used, such as the ProtParam tool from ExPASy (Expert Protein Analysis System), which can calculate the theoretical molecular weight and isoelectric point based on protein sequence.