Protein Assembly - Science topic
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I am purifying a 25KD protein with HIS label. The protein expression is large, but the proportion of the polymer is small. Meanwhile, the TEM photos taken by the polymer are also very vague.
I knocked out a gene which is expressed in skeletal muscle, one is hsp40 and another one is a co-chaperone of hsp90a1. Previous study showed that hsp90a1 is specially expressed in skeletal muscle, which involved in sarcomere protein assembly.
We have been carrying out an in situ proximity ligation assay (PLA) to detect the close association of two proteins on the plasma membrane of HEK293 cells. We find that the PLA signal increases when a third protein is present compared to its absence. However, we find that when oligomerisation is measured by STORM, we get evidence of association even in the absence of the third protein and no signal enhancement by the third protein.
My question: How quantitative is PLA? How well does PLA signal reflect the number of proteins in an assembled cluster? Has anyone done a quantitative comparison of PLA with other methods that measure protein associations, such as STORM?
I am trying to extract protein-complexes as intact as possible from the nuclei or even help them to get reassembled upon extraction. I do not know if the classic nuclear extraction protocols (e.g Dignam) could interrupt protein-protein interactions during the procedure, and if they do, I wonder if there is a protocol to help with the reassembly of protein-complexes --specifically transcription factors or machineries- the same as they used to be in the nucleus.
Thanks in advance
Amyloid and other protein filaments are associated with many neurodegenerative diseases and these often have associations with bound metal ions and reactive oxygen species. What is the current thought on appropriate therapeutic targets?