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Full length native protein (purified) corresponding to Cow Ubiquitin conjugated to Keyhole Limpet Haemocyanin (KLH).
Our Abpromise guarantee covers the use of ab19247 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use at an assay dependent concentration.|
|WB||1/1000 - 1/5000. Predicted molecular weight: 10 kDa.|
|IP||1/100. PubMed: 20103532|
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. ab19247 and ab8245 (loading control to GAPDH) were diluted 1/200 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging using the Licor Odyssey CLx.
Scrambled and lentivirus-stable infected SN4741 cells grown on coverslips were induced to differentiate under restrictive conditions for 24 h (pre-lethal stage), fixed and permeabilized with 0.1% Triton X-100 + 0.1% sodium citrate for 5 minutes. Cells were blocked with 10% donkey serum in PBS for 1 hour at 37°C and incubated over night at 4°C with rabbit polyclonal antibody against ubiquitin (ab19247, 1:100) and mouse monoclonal α-synuclein antibody (1:25). Samples were washed twice with PBS for 10 minutes before the addition of secondary antibodies at room temperature in the dark for 1 hour
Scrambled infected (control) cells show a diffuse staining pattern and do not show any inclusions at both permissive and restrictive temperatures (upper images). SKP1A-deficient cells developed multiple and perinuclear (bottom images, see arrows and insets) inclusion bodies 24 h after induction of differentiation, with characteristics of aggresomes. The reactivity of the aggregates to the different antibodies demonstrated a similar pattern as indicated by co-localized yellow immunostaining in the overlaid right-hand panel. No fluorescence was detected when the primary antibody was omitted. Each panel shows a representative picture of 10–15 views in two independent experiments.
Images top - bottom:
Scrambled 33°C/10% FCS,
SKP1A shRNA-1 33°C/10% FCS,
Scrambled 39°C/0.5% FCS,
SKP1A shRNA-1 39°C/0.5% FCS.