I got three bands in Western Blot for nicastrin (mol. wt. 110-120 kDa) after harvesting the protein from 4 breast cancer cell lines (MDA MB 231, MCF 7, T47D and ZR 75). The approximate size of the bands are 140, 120 (as expected) and 80 kDa and I used the nicastrin primary antibody from Sigma (Cat. No. N1660).
The appearance of the 3rd smallest band may reflect to the immature, non-glycosylated nicastrin (78.4 kDa). But what is the reason for the largest (and also thickest) band? Does it correspond to any pro-protein or precursor sort of molecule from which nicastrin might have been derived by way of post-translational cleavage (which results in truncated immature protein, which then undergoes further post-translational modification like phosphorylation or glycosylation to produce mature nicastrin)?
Hello, have you considered non-specific bands ? N1660 is a polyclonal antibody, which therefore recognize several epitopes, so that should not be surprising having several bands, and I would not try to draw conclusions on those extra bands because of this fact.
Try sonication before loading , it may help reducing background binding of teh antibody.
Thanks Alexis Varin and Anchit Khanna for your useful informations and suggestions.
- Badges
- Science topic