Importance of Sample Preparation and Choosing the Right Gel for SDS-PAGE
It is widely perceived that protein pre-cast or hand-cast gels are chemically inert and do not cause protein modifications during electrophoresis. However, multiple publications have reported chemical modification of amino acid side chains during separation of proteins by electrophoresis, including deamidation, aspartate–proline bond cleavage, methionine and tryptophan oxidation, and the Michael addition of sulfhydryl or amino groups to the double bond of acrylamide that is not polymerized into the gel matrix. These modifications can change the electrophoretic mobility of proteins resulting in blurred or multiple bands and are detrimental for downstream research applications. For example, mass spectrometry of the modified protein sample results in the predicted peptides appearing at several masses due to the diversity of unwanted modifications. Choosing the right gel chemistry for a specific research application can help minimize protein modification. For example, neutral pH Bis-Tris gels are recommended for samples with low abundance of target proteins or when downstream applications require high protein integrity (mass spectrometry, posttranslational modification or protein sequencing). Similarly, high molecular weight proteins (up to 500 kDa) can be optimally resolved by using Tris-Acetate gels whereas low molecular weight proteins (as low as 2.5 kDa) can be optimally resolved using Tricine gels. This webinar will highlight the different protein modifications occurring during electrophoresis, the importance of pH in sample integrity, how to choose the right gel chemistry for specific research applications and how to introduce the right new gel chemistry in your research workflow. Learning objectives How to choose the right pre-cast gel chemistry for your specific research application. Why Bis-Tris gel chemistry is ideal for post-translational modification applications. Why Tris-glycine gel chemistry is not ideal for all research applications.