All lanes : Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631) at 1/500 dilution

Lane 1 : HeLa cell lysate
Lane 2 : Jurkat cell lysate
Lane 3 : Human liver lysate
Lane 4 : Human liver membrane fraction lysate
Lane 5 : Human skeletal muscle lysate
Lane 6 : Human brain lysate
Lane 7 : Human kidney lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/50000 dilution

Performed under reducing conditions.

Additional bands at: 100 kDa (possible post-translational modification), 110 kDa (possible post-translational modification), 120 kDa (possible post-translational modification)


Exposure time: 20 minutes

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 staining human normal placenta. Staining is localized to the cytoplasm.
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 EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit 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.

Overlay histogram showing THP1 cells stained with ab25631 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab25631, 0.5µg/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. This antibody gave a positive signal in THP1 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

All lanes : Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631) at 1/500 dilution

Lane 1 : HEK293 cell lysate at 30 µg
Lane 2 : HepG2 at 30 µg
Lane 3 : THP-1 at 30 µg
Lane 4 : 3T3-L1 at 30 µl
Lane 5 : Mouse hepatocytes at 30 µl
Lane 6 : Rat hepatocytes at 30 µl

Secondary
All lanes : HRP conjugated Goat anti-Mouse IgG at 1/2000 dilution

Developed using the ECL technique.

Exposure time: 30 seconds

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.

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Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631) at 1/500 dilution + whole cell lysate prepared from THP-1 macrophages at 30 µg

Secondary
Goat anti-mouse IgG conjugated to HRP at 1/2000 dilution

Developed using the ECL technique.

Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds


Primary antibody incubated for 12 hours at 4°C.Gel running conditions: 12%Blocked with 5% milk for 1 hour at 25°C.

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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.

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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.