Cat. # | Size | Qty. | Price |
---|---|---|---|
13726S | 100 µl |
|
REACTIVITY | H R |
SENSITIVITY | Endogenous |
MW (kDa) | 23, 28 |
Source/Isotype | Mouse IgG1 |
Product Information
Application | Dilution |
---|---|
Western Blotting | 1:1000 |
Immunofluorescence (Immunocytochemistry) | 1:50 |
Flow Cytometry (Fixed/Permeabilized) | 1:50 |
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 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: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.
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 June 2020
Protocol Id: 262
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multiwell plates, chamber slides or on coverslips.
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
posted November 2006
revised December 2010
Protocol Id: 146
All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.
NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.
NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.
NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.
NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.
NOTE: Count cells using a hemocytometer or alternative method.
posted June 2005
revised June 2020
Protocol Id: 406
Human, Rat
Monoclonal antibody is produced by immunizing animals with recombinant protein encompassing the full-length of human PSMB8/LMP7 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).
Constitutively expressed core particle subunits PSMB5, PSMB7, and PSMB6 provide chymotrypsin-like, trypsin-like, and caspase-like activities, respectively (3). In immune cells involved in antigen presentation, these subunits are replaced by highly homologous, induced β-subunits to form the immunoproteasome (4,5).
Proteasome subunit beta type-8 (PSMB8, LMP7) is expressed as a proenzyme that is cleaved to form the mature PSMB8 (LMP7) immunoproteasome core particle subunit (6). Interferon-γ induces expression of PSMB8, which functionally replaces the PSMB5 core particle subunit in immunoproteasome processing of MHC class I-restricted peptide antigens (7). Research studies suggest that reduced PSMB8 expression or expression of the non-functional LMP7-E1 isoform may impair immunoproteasome assembly, and that PSMB8 deficiency results in reduced MHC class I molecule expression (8-10). Inhibition of PSMB8 in murine rheumatoid arthritis models attenuates disease indicators, suggesting that PSMB8 is a potential therapeutic target in the treatment of some proinflammatory autoimmune diseases (11). Mutations in the corresponding PSMB8 gene can cause an autoinflammatory syndrome known as CANDLE Syndrome (12).
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