![]() A protein that has the same isoelectric point as the pH of of the transfer buffer will have no net charge and thus will not migrate in an electric field.Isoelectric point of the protein is at or close to the pH of the transfer buffer In addition, excess methanol can tend to shrink or tighten a gel, thus inhibiting transfer of large molecular weight proteins. If protein precipitation is an issue, the transfer buffer can be supplemented with SDS (0.01% – 0.05%) to aid in solubility. This would reduce the transfer of large molecular weight proteins out of the gel. If the methanol concentration in the transfer buffer is too high, it can remove SDS from proteins and lead to protein precipitation in the gel.Switch to Immobilon®-PSQ transfer membrane.The membrane must be pre-wet with methanol the entire membrane should change uniformly from opaque to semi- transparent.Increase the methanol to 15 – 20%, especially for smaller molecular weight proteins. Methanol concentration in transfer buffer is too low to facilitate removal of SDS.Gel (top) and blot (bottom) show that equilibration improves transfer efficiency for Western blot. The entire membrane must be pre-wet with methanol the entire membrane should change uniformly from opaque to semi-transparent.Īir bubbles under membrane and between other layers in the stack.Membrane not uniformly wetted with methanol Poor Transfer of a Wide Range of Protein Sizes.Poor Transfer of Positively Charged Proteins.Poor Transfer of Large Molecular Weight Proteins (~ >80 kDa).Poor Transfer of Small Molecular Weight Proteins.High Background in Fluorescent Detection.Both are based on the same principles and differ only in the mechanical devices used to hold the gel/membrane stack and applications of the electrical field.Ĭlick on the Semi-dry or Tank Electrotransfer symptoms to read about the possible causes and remedies: The two commonly used electrotransfer techniques are tank transfer and semi-dry transfer. Related Resources: Brochures | Application NotesĮlectrotransfer refers to the standard procedure for transferring proteins from a polyacrylamide gel (SDS-PAGE) onto an Immobilon® PVDF transfer membrane.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |