Hello, I want to do EMSA with native PAGE to check protein-dna interactions.
The PIs of my proteins are between 8.1-8.5. I know that the pH of my buffer must be higher, so that the net charge is negative and the protein goes "downwards" to the anode. But do I have to adjust the pH (e.g. let's say 9.5) of everything? So separating gel, running buffer and loading dye? Or is the gel enough? I cannot find anything about running buffer and loading dye.
I my group we only did discontinous native gels so far, but in all recipes the pH of the stacking gel is around 6.8. Then my protein would run out of the gel, wouldn't it? Can I also change the pH of the stacking gel without changing the purpose of the stacking gel? I also found continuous native gels on the internet. Does that really work without getting a big smear?
In Native PAGE (Polyacrylamide Gel Electrophoresis), the pH value of each component plays a crucial role in ensuring the proper separation and migration of proteins based on their native charge and size. maintaining appropriate pH values for each component in Native PAGE is essential for preserving the native structure and charge of proteins, ensuring accurate separation and analysis. Any deviation from the optimal pH range can lead to protein denaturation, aggregation, or altered migration patterns, affecting the reliability and reproducibility of the results.
do not forget that for SDS PAGE there is SDS (that denatures and "cover " your protein and that you denatured the proteins by boiling + beta maercapto so the migration is not related to the pi of your protein; in some case the migration is not the migration expected from the MW of the protein but I do not know case of protein going backward on SDS page . the pH8.8 for separating gel and 6.8 will work
sorry I did not realize that your are doing native gels ... so my answer is not appropriate ;-(
- Badges
- Science topic
- Similar topics
- Medicine
- Physiology
- Biosignals
- Biomedical Signal Processing
- Statistical Analysis