Protein Assembly - Science topic

Jin Wang

Affinity purification of His-tagged fusion proteins is the most common application of metal chelated carriers in protein biology research. Check again whether or not the cleaning method suits your task. It is better to use Cryo TEM to study the shape of the protein, as Alan writes. Water retains its protein form. In TEM she disappears.

If you haven't already, the first thing you should do is confirm successful knock out of your gene/s. If a specific antibody is available, confirm the absence of protein by western blot or, even better, confirm its absence in skeletal muscle using IHC. Can you detect evidence of nonsense-mediated decay by in situ hybridisation?

If the knock out is successful, then you can explore potential reasons for the lack of phenotype, where one might be expected. One possibility is compensation by paralogous genes with similar function. Can you detect an increase in mRNA transcripts which might be compensating? Are they expressed in muscle?

Another possibility is that you are missing a phenotype. How are you looking for evidence of poor sarcomere assembly? You should, for example, stain for a range of myofibrillar proteins (e.g. MyHC, actin, alpha-actinin, titin.... etc)...

Cheers,

Mike.

Not sure. By it's nature PLA should be (at least in principle...) dependent on conformation of target protein assembly that will affect binding of antibodies and therfore geometry and amplification rates. Even if AB distance and frequency does not change if C is added, ABC complex could be (PLA) chemically different (either higher or lower) if conformation is more accessible to PLA ingredients.

Hi James. Thanks for the reply. Since I am looking for TF-complexes, cross-linking might fix them on the chromatin or even change their native conformation; while, I would like to extract the native complexes from nuclei and apply them in other assays and I am looking for protocols with least disturbance of protein complexes.

I totally agree though that complexes in more concentrated extracts have a much higher chance to stay intact.

John

Too often alternate assemblies of homo-multimeric proteins are perceived as properly assembled and mis-assembled. This is not always the most productive view. In some cases alternate assemblies simply reflect alternate physiologically relevant alternate functions and the proteins fluctuate between alternate assemblies in response to cellular needs. The alternate functions can be an "on" state vs an "off" state or two alternate moonlighting functions. We have published extensively on this topic and have established a variety of precedents using the porphobilinogen synthase system, where an octamer is the "on" state, a hexamer is the "off" state. In this case, allosteric ligands bind to multimer-specific small molecule binding sites and modulate the activity. A similar approach is being considered for phenylalanine hydroxylase, where stabilizing the "on" assembly is a legitimate therapeutic target. I believe that multimer-specific small molecule binding sites on alternate protein assemblies constitute an under-explored therapeutic target with great potential for future growth.