Mouse, Rat, Rabbit, Human, Saccharomyces cerevisiae, Potato Predicted to work with:
a wide range of other species
Full length protein (dinitrophenylated proteasomes).
Reacts with the peptide sequence TVWSPQGRLHQVEYAMEA encompassing the prosbox I motif common to alpha type subunits, although not necessarily identical in all. In general this motif is phylogenetically preserved.
Human placental proteasome.
Dilute antibody to working dilution in PBS , pH 7.2 - 7.4. Store at 4°C and use within 1 month. Do not freeze.
This antibody reacts with six different alpha type subunits:- alpha 1, 2, 3, 5 , 6 and 7.
Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 2µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use a concentration of 1 - 5 µg/ml.
1/1000. Detects a band of approximately 29 kDa (predicted molecular weight: 29 kDa).
Use at an assay dependent concentration.
Is unsuitable for IP.
The proteasome is widely recognised as the central enzyme of non lysosomal protein degradation. It is responsible for intracellular protein turnover and it is also critically involved in many regulatory
processes and, in higher eukaryotes, in antigen processing. The 26S proteasome is the key enzyme of the ubiquitin/ATP dependent pathway of protein degradation. The catalytic core of this unusually large complex is formed by the 20S proteasome, a barrel shaped structure shown by electron microscopy to comprise of four rings each containing seven subunits.
Based on sequence similarity, all fourteen 20S proteasomal subunit sequences may be classified into two groups, alpha and beta, each group having distinct structural and functional roles. The alpha subunits
comprise the outer rings and the beta subunits the inner rings of the 20S proteasome. Observations of the eukaryotic proteasome and analysis of subunit sequences indicate that each ring contains seven different subunits (alpha7/beta7/beta7/alpha7) with a member of each sub family represented in each particle. Each subunit is located in a unique position within the alpha or beta rings.
20S Proteasomes degrade only unfolded proteins in an energy independent manner, whereas 26S proteasomes degrade native and ubiquitinylated proteins in an ATP dependent manner. The native protein substrates are recognised by subunits, some with ATP binding sites, of the outer 19S caps of the 26S proteasome.
ICC/IF image of ab22674 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab22674, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Overlay histogram showing HepG2 cells stained with ab22674 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab22674, 2µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1](ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.