i am performing western blotting since past 4 months, but till now i am not confident with the results i am getting because of the following reasons-
1] previously i use to use 1.5M TRIS pH 6.8 and 1.5M TRIS pH 8.8 for stacking and running gel respectively but every time the gel was having some fibrous projections at the wells which use to prevent the proper loading, as a result one of the seniors here recommended to use 1M TRIS pH 7.4 and 1M TRIS pH 8.9 for stacking and running gels respectively. the gels are fine now but i dont know weather it is a good way or not.
2] i always check bactin for calibrating if equal amount of protein is been loaded and followed by other antibodies. But what i observe recently is, even after removing the prior ab by streaping solution, and performing all the steps for further ab i am getting same kind of band, with same intensity and everything same.
i reuse one blot atleast at a time diff of 12 hours to make sure that the band intensity is not repeated.
I will be very thankful for your kind comments on these kind of problems.