|H M R Mk||Endogenous||47||Rabbit IgG|
Western blot analysis of extracts from various cell lines using PSMC2 (D5T1T) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). OVCAR8 cells are reported to express low levels of PSMC2 (7).Learn more about how we get our images.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human PSMC1 protein (hPSMC1-Myc/DDK; +), full-length human PSMC2 protein (hPSMC2-Myc/DDK; +), full-length human PSMC3 protein (hPSMC3-Myc/DDK; +), full-length human PSMC4 protein (hPSMC4-Myc/DDK; +), full-length human PSMC5 protein (hPSMC5-Myc/DDK; +), and full-length human PSMC6 protein (hPSMC6-Myc/DDK; +), using PSMC2 (D5T1T) Rabbit mAb (upper) and DYKDDDDK Tag Antibody #2368 (lower).Learn more about how we get our images.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 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.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
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 October 2016
Protocol Id: 10
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
PSMC2 (D5T1T) Rabbit mAb recognizes endogenous levels of total PSMC2 protein. This antibody does not cross-react with other AAA-ATPase subunits of the 19S proteasome regulatory particle.
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human PSMC2 protein.
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). PSMC2 (RPT1, MSS1) is a AAA-ATPase subunit of the 19S/PA700 RP. Research studies have shown that PSMC2 is associated with several components of the basal transcriptional machinery suggesting that PSMC2, in addition to participating in proteasome-dependent degradation of proteins, may also play a role in gene transcription (6). More recently, it has been shown that numerous human cancer cell lines have reduced PSMC2 expression resulting from loss of PSMC2 copy number loss and display a strict threshold requirement for PSMC2 levels in order to sustain a proliferative advantage (7).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc.
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|14395S||100 µl (10 western blots)||$ 255.0|