I am interested in performing Ubiquitination analysis from whole lysates, however most protocols enriching for Ubiquitinated peptides using the KGG motif (enriched using IP for KGG peptides) has been performed on very high starting materials. (5mg or more).
Has anyone tried scaling it down to rougly 100 microgram input material?
the reason that mg amounts is recommended is due to the small fraction of proteins carrying ubiquitination. if ~0.1% of the proteins are ubiquitinated, then 5mg will result in ~5ug of ubiquitinated proteins. taking into account losses due to processing, you may end up with ~50% of that amount.
that means that out of 100ug, you'll end up with 100ng - not too much. it's possible, but may not show as many as you could see with more starting material.
Hi David. many thanks for the answer. I get the point that scaling down will result in much smaller amount of ubiquitinated peptides being obtained.
Its interesting that you mention 0.1%, where do you get this number from?
Best
nikhil
0.1% is not a hard number. the most common PTM in the cell is phosphorylation and that is about 1% of the peptides in the cell. other PTMs may take much less then that. like many other PTMs, ubiquitination is also dependent on the situation of the tissue (tumor, apoptosis, etc...)
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