Ubiquitin is a highly conserved regulatory protein expressed in all eukaryotic tissues. Originally this protein was called: Ubiquitous Immunopoietic Polypeptide. Its function is labeling of proteins for degradation through ubiquitin proteasome system (UPS).
Source of positive control, ubiquitin protein: full length recombinant Arabidopsis thalianaubiquitin, ubq11, with N-terminal 1XHis-3XHA-tag; locus accession: At4g05050
immunogen
does not apply
host
does not apply
quantity
50 µl
reconstitution
liquid
storage
store protein standard at -20°C; Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tubes.
Protein is provided in Laemli SDS loading buffer and should be heated up in 98°C for 5 minutes before loading on the gel.
APPLICATION INFORMATION
recommended dilution
positive control: a 2.5-5 μl load per well is optimal for most chemiluminescent detection systems.
expected | apparent MW
10-12 kDa
not reactive in
no confirmed exceptions from predicted reactivity currently known
additional information
Concentration: 0.8 µg/µl. Recommended load per gel to allow Coomasie staining and western blot detection is 2.5-5 µl.
1x SDS Loading Buffer contains:
50 mM Tris-HCl pH 6.8
2% SDS
10% glycerol
1% β-mercaptoethanol
12.5 mM EDTA
0.02% bromophenol blue
selected references
to be added when available
application example
5 μg of recombinant protein from Phytophora sp. (1), human (2), Arabidopsis thaliana His-tagged ubiquitin (3),Arabidopsis thaliana His-tagged SUMO protein (4), was separated on 15% PAA gel and blotted on PVDF membrane. Filters were blocked in 5% milk for 1h, incubated with 1: 10 000 anti-ubiquitin antibody (1h), followed by incubation with 1: 15 000 secondary anti-rabbit antibodies(1h) coupled with HRP and visualization (10 seconds exposure) with standard ECL.
Technical note: It is very difficult to detect ubiquitin monomers in total cell extracts due to a great abundance of poly and multi-ubiquitinated proteins. Recommended is size separation of protein extracts before gel electrophoresis focused on good resolution of region between 6-10 kDa.