UBQ11 | Ubiquitin protein (positive control)
AS10 307S | protein | positive control
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positive control: a 2.5-5 μl load per well is optimal for most chemiluminescent detection systems.
|Expected | apparent MW||
|Not reactive in||
no confirmed exceptions from predicted reactivity currently known
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:
to be added when available
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.
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