Immuno-precipitation & Western Blots often suffer from heavy/light chain blotting, contamination, and ongoing interferences. This can prevent obtaining biologically-relevant data in a given experimental model, especially when the target of interest has a size similar to the IgG molecules that may remain. Getting rid of these “artificial” bands can be tricky. In this post, we’ll be taking a look at the robustness of the TrueBlot® products for facing these issues.
Western blotting (WB) is an antibody-based method enabling sensitive detection of a protein of interest from other proteins present in biological samples. Detection is made after an initial separation step of the proteins according to their molecular weight, by PolyAcrylamide Gel Electrophoresis (PAGE). After immobilization onto a nitrocellulose, nylon or PVDF membrane, the protein of interest is revealed by its interaction with appropriate primary and secondary antibodies, followed by enzymatic (or fluorescent) methods.
Here are 2 tactics, that we’ve all been taught to keep in mind, but which will really help you towards designing successful WB protocols. A big thank you to Dana and Camillo (Rockland Immunochemicals) for their support here. [Read more…]
When preparing a protocol for Western blotting phosphorylated proteins, a few essential points should be taken into consideration to obtain accurate immunoblotting results. Your regular WB protocol, such as the WB protocol edited by tebu-bio’s antibody users, can be fine-tuned according to this “key” post-translational protein modification.
We’ve put together 5 tech tips, which are sure to support you in designing your phosphorylated protein Western blot:
- Check phospho-specificity of primary antibodies used in Western blot
- Pamper your phospho-proteins in your samples
- Use blotting buffers compatible with phosphorylation studies by Western Blot
- Enrich your samples to detect low abundance proteins
- Stripping and re-probing your Western Blot membrane for using another antibody
Download the Guide
“5 Tips for Phosphorylated Western Blot”