SIGMA-ALDRICH
ProteoPrep® Reduction and Alkylation Kit
PROTRA-1KT
I work on diabetic and obesity animal models. In this study, I have given various natural as well as synthetic agents to those animals to check the progress of the diseased condition. After the long duration of intervention study, I scarified the animals and checked the important proteins expression in both control and experimental animals. As part of this study, I have done 2D electrophoresis. Initially, I found poor separation of proteins then came to know that Disulfide bond breaking is critical. I selected this product by myself and tried it.
2D gel electrophoresis.
Diabetic rat liver.
Prepared protein (1mg/ml) sample in the sample buffer and diluted with additional sample buffer which was loaded onto IPG strips. Adjusted the sample pH to 9.0 by adding 15 µl of the alkylation buffer. Vortexed the sample to that Added 12.5 µl of TBP for a final concentration of 5 mM. Again mixed and incubated at RT for 30 min. To the sample 15 µl of 0.5 M iodoacetamide was added and mixed well by vortexing. Incubate at RT for 1 hr. Added an additional 12.5 µl of TBP to quench any unreacted iodoacetamide. Mixed well by vortexing. Incubate at RT for 15 min. Centrifuge at 12,000 x g for 8 min at RT. Adjusted the protein sample to final volume with sample buffer and that was loaded onto Strip.
Prepare iodoacetamide freshly, is very unstable in solution.
Observed the increased 2D spot resolution increased efficiency of protein samples when compared with untreated samples.
Better to follow the instructions strictly.
Highly useful for 2D and MS applications.
Nothing
This kit is good to use for high quality results.