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The details of the immunogen for this antibody are not available.
Our Abpromise guarantee covers the use of ab25631 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use 0.5-1µg for 106 cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
|IHC-Fr||Use at an assay dependent concentration.|
|ICC||Use at an assay dependent concentration.|
|WB||Use at an assay dependent concentration.
In our hands milk blocking is gives superior results to BSA blocking for this product.
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|IHC-FoFr||Use at an assay dependent concentration. (PMID 19837699).|
Along with THP-1 macrophages, other human cell lines were loaded, and the 110 kDa mature band for LAMP2 was detected in all the samples. On the other hand, mouse and rat cells were negative. The antibody works really good on human samples, detecting a single 110 kDa band but it's not suitable to use for mouse or rat samples.
This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system under denaturing, reducing conditions. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. After transfer, the membrane was blocked for an hour in 3% milk before being incubated overnight at 4°C with mouse monoclonal [H4B4] to LAMP2 (ab25631; diluted 1:5000). Antibody binding was detected using peroxidase labelled goat anti-mouse IgG (ab97040; diluted 1:50000) for an hour at room temperature and visualised using ECL development solution.
ab25631 at 0.5µg/ml staining human HEK cells by immunocytochemistry. The cells were fixed with paraformaldehyde and incubated with the antibody for 1 hour. Bound antibody was detected using a goat anti-mouse IgG Alexa-Fluor ® 568. In the confocal image ab25631 labelling in red shows a distribution consistent with the location of lysosomes. Blue nuclear counterstain is present.
This image is courtesy of an Abreview submitted by Randal Moldrich on 31 March 2006.
ab25631 staining LAMP2 in human THP-1 cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100 and blocking with 1% BSA at 220C for 2 hours was done. Samples were incubated with primary antibody (1/400: in PBST and 1% BSA) for 16 hour at 40C. An Alexa Fluor ® 568-conjugated goat polyclonal to mouse IgG was used at dilution at 1/500 as secondary antibody. Merge figure shows the bright field image and fluorescent image combined.
Western blot analysis of Rhesus monkey primary retinal pigmented epthelium whole cell lysate and HeLa cell whole cell lysate (20µg/lane) labelling LAMP2 with ab25631 at 1/1000. An alkaline phosphatase-conjugated rabbit anti-mouse IgG was used as the secondary antibody.
Flow Cytometry analysis of Human T lymphocyte cell line Jurkat labeling LAMP2 with ab25631 at 1 μg/106 cells dilution (purple). A Goat Anti-Mouse IgG1, Human ads-FITC was used as the secondary antibody. Grey - Isotype Control, Mouse IgG1-UNLB, followed by Goat Anti-Mouse IgG1, Human ads-FITC.