Should I use 1D or 2D SDS PAGE?
2D PAGE is a classic method for separating and visualizing many proteins from complex proteomics samples. It allows you to assess the purity of a sample, which is very useful before performing mass spectrometry analysis on samples that rely on relatively high chromatographic purity (>80%), such as intact mass determination or peptide mapping. However, 2D PAGE also has some drawbacks. For example, you rarely observe hydrophobic membrane proteins because they precipitate during IEF. Therefore, you are likely to only observe proteins within the pI range of the gel, typically with a pH of 4-7 or 3-10, and the molecular weight range of proteins in the gel is about 10-130 kDa. Therefore, for large hydrophobic proteins, it is better to use 1D SDS PAGE. The main reason is that you can dissolve proteins in 1D SDS PAGE buffer containing 0.1% SDS. Beyond that, the gel has no pI limitation, and the MW range can be as high as 1000 kDa. Another possibility is solution digestion for protein mixtures. This method is an alternative to running the gel and cutting out protein bands of interest before in-gel digestion and identification of protein IDs for each band. Typically, you identify hundreds or even thousands of proteins via LC-MS/MS and subsequent database searches in Swiss-Prot/UniProtKB.
How to order?
