Hi,
I am testing a commercial importin antibody generated in rabbit with drosophila fly lysates. And i get strong bands of wrong size (unspecific bindings) whereas i cant find any bands of correct size. I tried with human and mouse cell lysates and the antibody works fine for them, but not with the fly lysate. Has anyone had similar kind of experience with this antibody and how did you solve it?
Any suggestions would be appreciated. Many thanks.
Hi there,
Thank you for the materials.I think my lysate preparation procedure is fine because by following the same protocol for human and mouse cell lysate, i got my desired protein's band. However, it doesnot work with the fly lysate only. I have tried using two different companies antibodies to detect protein of my interest,none worked.
1. Are you sure the level of your protein of interest in flies is high enough to be detected?
2. Do you know what is the antigen epitope of your antibody would bind to? Have you compared the amino acid sequence of your protein of interest across the species that you have tested on? The amino acid discrepancy across different species, if there is any, would not allow antibody binding if it happens to span across the antigen epitope.
3. Are you running native or SDS PAGE? Sometimes the post-translational modification would mess up the epitope and disable the antibody on native gel. The apparent size of the protein would also be different if you run it on native gel.
4. How much does the molecular weight of the non-specific band deviate from the expected size? Sometimes the post-translational modification, for example glycosylation and phosphorylation, would change the molecular weight of the protein and therefore the size of the protein band.
Good luck.
- Badges
- Science method