A biologist ran a Western blot analysis for 2 compounds of mine in SK-N-BE(2) neuroblastoma cell line and gave me the attached 2 gel images one for p-Tau (p-S396) (https://www.scbt.com/p/p-tau-antibody-phf-13 ) and another for Tau 46 (https://www.scbt.com/p/tau-antibody-tau-46 ). He highlighted 2 bands (appearing as a doublet) in Tau46 and I'm not sure why is it appearing as 2 bands, is this due to phosphorylation dependent mobility shift, and accordingly the upper band is phosphorylated while the lower one is not, or what can the explanation be? And when I calculate the p-tau/ total tau ratio from the densitometric analysis I ran for those 2 bands, which one should be the one used as the total tau, is it the lower band or should I sum the 2 bands, or how is it calculated?
In addition, the same happened in case of testing p35 protein using this antibody (https://www.scbt.com/p/p35-antibody-b-1 ), he highlighted 2 bands and labelled them as p35, and I'm not sure why? I found that phosphorylation dependent mobility shift was reported before for p35 in this article https://www.sciencedirect.com/science/article/abs/pii/S0169328X02004096 what do the 2 bands represent? is the upper one phosphorylated form and the lower one is unphosphorylated?
The first lane in each gel is DMSO used as a control.
Any help in clarifying this issue would be highly appreciated!
Optimize your western blot conditions to reduce unspecific bands. Several primary antibodies work great at a 1:1000 dilution; try diluting your antibody in 5% BSA to reduce non-specific binding. If the background persists, increase the blocking time (e.g., from 1 h to overnight at 4°C). You may use a more stringent lysis buffer (e.g., RIPA buffer instead of NP-40) to reduce unspecific interactions.
Kind regards
Your western blots are full of non-specific bands. Only the band for your protein of interest should be present. You need to optimise your WB protocol as the bands at the expected molecular weight may not even be the protein you're interested in.
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