In Northern blot, we normally use either 2*SSC+0.1% SDS or 0.1*SSC+0.1% SDS in washing step. Does anyone know what's the function of the SDS in the washing buffer?
Thank you in advance
SDS is ionic denaturing detergent. Hot SDS buffer is often used when the proteins need to be completely solubilized and denatured.SDS in the buffer helps keep the proteins linear. Glycine is an amino acid whose charge state plays a big role in the stacking gel.
The anionic SDS is a very commonly used and effective surfactant in solubilizing most proteins. It disrupts non-covalent bonds within and between proteins, denaturing them, and resulting in the loss of their native conformation and function. SDS binds to a protein with a ratio of 1.4:1 w/w (corresponding to about one SDS molecule per two amino acids), masking the charge of the protein. Thus SDS adds an overall negative charge to all proteins in the sample regardless of their isoelectric point (pI). Once bound by negatively charged SDS molecules the proteins can be separated based on size. That is a big reason for the wide use of SDS polyacrylamide gel electrophoresis (SDS-PAGE) for separating and studying proteins. Usually, for complete cell lysis in the presence of SDS, a sample must be sonicated or sheared (e.g., passed through a 19G needle) several times to ensure DNA degradation. SDS cannot be used when active proteins are required or when protein-protein interactions are being studied because both of these are disrupted by the SDS. When working with SDS it is important to know that SDS precipitates at low temperatures, and this effect is enhanced in the presence of potassium salts. This phenomenon can sometimes be exploited to remove SDS from a protein sample.
- Badges
- Science method