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Full length protein corresponding to Ubiquitin conjugated to Keyhole Limpet Haemocyanin (KLH). Mouse monoclonal [Ubi-1] to Ubiquitin was raised against purified ubiquitin conjugated with glutaraldehyde to keyhole limpet hemocyanin.
This product was changed from ascites to tissue culture supernatant on 17th May 2016. The following lots are from ascites and are still in stock as of 17th May 2016: GR159581-2, GR159581-12, GR159581-13. Lot numbers higher than GR159581-13 will be from tissue culture supernatant. Please note that the dilutions may need to be adjusted accordingly.
Our Abpromise guarantee covers the use of ab7254 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-P||Use a concentration of 1 - 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|WB||1/100 - 1/5000.|
|ELISA||Use at an assay dependent concentration.|
IHC image of ab7254 staining in mouse alzheimer brain formalin fixed paraffin embedded tissue section, using MOM detection kit, ab127055. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab7254, 5µg/ml, for 15 mins at room temperature. DAB was used as the chromogen (ab103723). The section was counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Immunohistochemical staining of ubiquitin in a 7 months-old MPS IIIA mouse brain (PFA fixed) using ab7254.
Sections, 40 µm thick, were cut sagittally on a vibratome. Slices were permeabilized in 1% triton X-100/PBS for 1 hour at room temperature. The samples were placed on vectabond-coated glass slides and dried overnight. They were then rehydrated in PBS and washed in ice-cold methanol for 10 min.
The slices were washed in 0.3% H2O2 in methanol at room temperature and incubated sequentially with 0.5% normal donkey serum, ab7254 and biotin-labeled F(ab')2 donkey secondary antibody in Tris-buffered saline (pH 7.5) containing 2% BSA and 0.02% Tween 20. The signal was detected with an ABC kit, visualized with diaminobenzidine and counterstained with hematoxylin.
ab7254 staining Ubiquitin in murine myocytes by Immunocytochemistry/ Immunofluorescence. Isolated myocytes were treated with 10µM FCCP for 1 hour, or untreated as the control. Cells were then fixed in formaldehyde, permeabilized using 1% Triton X-100, then blocked with 10% horse serum for 1 hour at room temperature, incubated with ab7254 at a 1/200 dilution. The secondary used was an Alexa-Fluor 488 conjugated goat anti-mouse polyclonal (ab150113), used at a 1/500 dilution.Results show that with FCCP treatment, Ubiquitin is recruited onto mitochondria.
This image is courtesy of an anonymous Abreview
Human normal placenta (ab29745). Staining is localised in the cytoplasm and in the nuclei. Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus, at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for mouse for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
ab7254 staining Ubiquitin in Rat h9c2 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 1% Triton X-100 and blocked with 5% serum for 1 hour at room temperature. Samples were incubated with primary antibody (1/200 in 5% Goat serum ab7481) for 1 hour at room temperature. An Alexa Fluor® 488-conjugated Goat anti-mouse IgG polyclonal (ab150113) (1/400) was used as the secondary antibody.
Image courtesy of an anonymous Abreview.HeLa cells were co-transfected with a plasmid expressing a target protein together with Ubi expressing vector for 24 hours and either left untreated (Contr) or were treated with 10 µM MG-132 for 6 hours (+MG132). Then the protein of interest was pulled down using Flag agarose beads and and probed with ab7254 at a 1/2000 dilution. The secondary used was an Alexa-Fluor 680 conjugated goat anti-mouse polyclonal used at a 1/10000 dilution. The protein is known to be degraded through proteasome.