Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human PSMC1 (hPSMC1-Myc/DDK; +), full-length human PSMC2 (hPSMC2-Myc/DDK; +), full-length human PSMC3 (hPSMC3-Myc/DDK; +), full-length human PSMC4 (hPSMC4-Myc/DDK; +); full-length human PSMC5 (hPSMC5-Myc/DDK; +), or full-length human PSMC6 (hPSMC6-Myc/DDK; +), using PSMC5/TRIP1 Antibody (upper) and DYKDDDDK Tag Antibody #2368 (lower).
Western blot analysis of extracts from various cell lines using PSMC5/TRIP1 Antibody.
|REACTIVITY||H M R Mk|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
PSMC5/TRIP1 Antibody recognizes endogenous levels of total PSMC5 (TRIP1) protein. This antibody does not cross-react with other AAA-ATPase subunits of the 19S proteasome regulatory particle.Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
D. melanogaster, Zebrafish, Pig
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human PSMC5 (TRIP1) protein. Antibodies are purified by protein A and peptide affinity chromatography.
The 26S proteasome is a highly abundant proteolytic complex involved in the degradation of ubiquitinated substrate proteins. It consists largely of two sub-complexes, the 20S catalytic core particle (CP) and the 19S/PA700 regulatory particle (RP) that can cap either end of the CP. The CP consists of two stacked heteroheptameric β-rings (β1-7) that contain three catalytic β-subunits and are flanked on either side by two heteroheptameric α-rings (α1-7). The RP includes a base and a lid, each having multiple subunits. The base, in part, is composed of a heterohexameric ring of ATPase subunits belonging to the AAA (ATPases Associated with diverse cellular Activities) family. The ATPase subunits function to unfold the substrate and open the gate formed by the α-subunits, thus exposing the unfolded substrate to the catalytic β-subunits. The lid consists of ubiquitin receptors and DUBs that function in recruitment of ubiquitinated substrates and modification of ubiquitin chain topology (1,2). Other modulators of proteasome activity, such as PA28/11S REG, can also bind to the end of the 20S CP and activate it (1,2).
The base of the eukaryotic proteasome 19S/PA700 RP contains six AAA-ATPase subunits (PSMC1-PSMC6) that bind directly to the 20S CP α-ring. These 19S RP ATPases are thought to assemble into a heterohexameric, pore-like structure that forms part of the substrate translocation channel. Energy derived from ATP hydrolysis by the AAA-ATPases is utilized for substrate unfolding and translocation, which is required for degradation of ubiquitinated folded proteins within the central chamber of the 20S CP formed by β-subunits (3-5). Thyroid hormone receptor-interacting protein 1 (PSMC5, TRIP1) is a 19S AAA-ATPase subunit involved in the negative regulation of gene transcription. Recruitment of PSMC5 to liganded VDR (6) and RARγ2 (7) facilitates their degradation via the proteasome.
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