Anti-LAMP2 antibody [H4B4] - Lysosome Marker

9 product images

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  This image is of an Abreciew submitted by Daniel Rodriguez.

  Western blot - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  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.

  All lanes: Western blot - 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.

  Predicted band size: 45 kDa

  Exposure time: 30s

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  This image was kindly supplied by Daniel Rodriguez by customer review

  Western blot - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

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

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

  All lanes: whole cell lysate prepared from THP-1 macrophages at 30 µg

  Secondary

  All lanes: Goat anti-mouse IgG conjugated to HRP at 1/2000 dilution

  Developed using the ECL technique.

  Predicted band size: 45 kDa

  Observed band size: 110 kDa

  Exposure time: 30s

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  Western blot - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  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 (Goat Anti-Mouse IgG H&L (HRP) preadsorbed ab97040; diluted 1:50000) for an hour at room temperature and visualised using ECL development solution.

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

  Lane 1: HeLa cell lysate at 20 µg

  Lane 2: Jurkat cell lysate at 20 µg

  Lane 3: Human liver lysate at 20 µg

  Lane 4: Human liver membrane fraction lysate at 20 µg

  Lane 5: Human skeletal muscle lysate at 20 µg

  Lane 6: Human brain lysate at 20 µg

  Lane 7: Human kidney lysate at 20 µg

  Secondary

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

  Performed under reducing conditions.

  Predicted band size: 45 kDa

  Observed band size: 100 kDa, 110 kDa, 120 kDa

  Exposure time: 20min

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  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.

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  Flow Cytometry (Intracellular) - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  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) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control 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.

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  This image is courtesy of an anonymous customer review

  Western blot - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  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.

  All lanes: Western blot - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  Performed under reducing conditions.

  Predicted band size: 45 kDa

  Observed band size: 110 kDa

  Exposure time: 1s

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  Flow Cytometry (Intracellular) - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  Intracellular 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.
  

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  Immunocytochemistry/ Immunofluorescence - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  LAMP2 Immunocytochemistry/ Immunofluorescence staining using mouse Anti-LAMP2 antibody

  Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling TRPML1/MG-2 with Anti-TRPML1/MG-2 antibody [EPR28651-127] ab323642 at 1/100 (5.07 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 1000 2ug/ml dilution (Green).

  Confocal image showing lysosomal staining in Neuro-2a cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
  
  Low expression: F9

  ab25631 Anti-LAMP2 mouse monoclonal antibody - Lysosome Marker was used to counterstain tubulin at 1/50 2ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

  Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/ 1000 2ug/ml dilution.

  -ve control 1: Anti-TRPML1/MG-2 antibody [EPR28651-127] ab323642 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.
  -ve control 2: ab25631 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.

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  Immunocytochemistry/ Immunofluorescence - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (ab25631)

  LAMP2 Immunocytochemistry/ Immunofluorescence staining using mouse Anti-LAMP2 antibody

  Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized PC-3 (human prostate adenocarcinoma epithelial cell) cells labelling TRPML1/MG-2 with Anti-TRPML1/MG-2 antibody [EPR28651-127] ab323642 at 1/100 (5.07 ug/ml) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 1000 2ug/ml dilution (Green).

  Confocal image showing lysosomal staining in PC-3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
  
  Low expression: Ramos

  ab25631 Anti-LAMP2 mouse monoclonal antibody - Lysosome Marker was used to counterstain tubulin at 1/ab25631 50 2ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

  Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 1000 2ug/ml dilution.

  -ve control 1: Anti-TRPML1/MG-2 antibody [EPR28651-127] ab323642 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) antibody at 1/1000 dilution.
  -ve control 2: ab25631 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.