This document introduces the effects of proteins and buffers on staining, denaturation, and electrophoresis when analyzing proteins with Agilent P200 ScreenTape
This document serves as a technical overview for analyzing proteins using the Agilent P200 ScreenTape and 2200 TapeStation system. It details how various protein properties, such as amino acid composition and modifications, can impact the staining reaction and overall analysis results. The text also highlights the variability introduced by different buffer compositions and additives used in protein preparation.
| Staining reaction optimal pH range | pH 8-10 |
|---|---|
| P200 pH buffer pH | pH 9 |
| Urea compatibility limit | 8M |
| Guanidine compatibility limit | 600 mM |
| Salt concentration limit for migration | 500 mM |
| Triton X-100 compatibility limit | 1% |
| Acetonitrile compatibility limit | 20% |
| Methanol compatibility limit | 20% |
| Ethanol compatibility limit | 20% |
The staining reaction occurs optimally between pH 8-10 as the amine group needs to be deprotonated to be available to the dye.
Guanidine has low compatibility at 600 mM because it causes SDS precipitation. It is recommended to dilute guanidine to below 600 mM or use urea instead.
High salt levels retard the dye front, which can affect sizing accuracy. Levels above 500 mM begin to retard the internal 10 kDa marker.
Care must be taken when pipetting small volumes of high viscosity samples to ensure reproducibility of analysis.