Problem- same bands every time

[thebadman]

Hello
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.

[protolder]

Hola, for the first question if the changes were checked before by your supervisor and you are satisfied with the results, probably were good, don´t mind. The second seems that the stripping isn´t not effective at all , because it´s extrange that the blocker or the second ab reacts with any band. I´m enought sure that if after blocking you incubate for first time the WB with the second ab and developes it no band you will see so the first ab strongly bound to the persistent bands, so it would be necessary to do a more efficient stripping with a bit more temperature and time. I used 20 min at 50ºC with rocking, and probably 25x55 could run, unless some of the trasferred protein could be unbounded. Buena suerte

[thebadman]

thanks for your comments, i generally perform stripping at 37 degrees for 30 mins. I hope it would be better to check if the stripping is happening correctly by -
stripping--> washing --> secondary ab -->washing -->substrate and anti-antibody.
if there is any un removed primary ab then it should show band. Please let me know your comments.

[protolder]

Hola, I thing that this T is quite low. Try to do at 50º 20min saking. To check is the stripping is effective , after washing stripping sol, first blocking, washes- 2nd anntibodi-HRP. washes developpimg. Buen día

[thebadman]

thanks a lot, i will try to perform this.