Anti-LAMP2 antibody [H4B4] - Lysosome Marker

• WB

AbReview16766****

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

Predicted band size: 45 kDa

true

Exposure time: 30s

This image is of an Abreciew submitted by Daniel Rodriguez.

• Flow Cyt (Intra)

Unknown

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) (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.

• Flow Cyt (Intra)

Supplier Data

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.

• IHC-P

Unknown

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (AB25631)

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

-ve control 1 : ab323642 at 1/100 dilution, followed by 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 ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (AB25631)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized PANC-1(human pancreatic epithelioid carcinoma epithelial cell); Raji (human Burkitt's lymphoma B lymphocyte) cells labelling TMEM106B with ab325555 at 1/500 (0.998 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing lysosome staining in PANC-1 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).

Negative control : Raji.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab25631 Anti-LAMP2 mouse monoclonal antibody - Lysosome Marker was used to counterstain lysosomes at 1/200 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at a 1/1000 dilution (Magenta).

-ve control 1 : ab325555 at a 1/500 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab25631 at a 1/200 dilution followed by ab150081 at a 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (AB25631)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Wildtype A549 (wildtype human lung carcinoma epithelial cell); TMEM106B KO A549 (TMEM106B knockout A549), ab273711 cells labelling TMEM106B with ab325555 at 1/500 (0.998 µg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing lysosome staining in wildtype A549 cells (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).

ab25631 Anti-LAMP2 mouse monoclonal antibody - Lysosome Marker was used to counterstain lysosomes at 1/200 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 dilution (Magenta).

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (AB25631)

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

-ve control 1 : ab323642 at 1/100 dilution, followed by 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 ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (AB25631)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized 3T3-L1 (mouse embryonic fibroblast) cells labelling TMEM106B with ab325555 at 1/500 (0.998 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing lysosome staining in 3T3-L1 cells (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).

ab25631 Anti-LAMP2 mouse monoclonal antibody - Lysosome Marker was used to counterstain lysosomes at 1/200 dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at a 1/1000 dilution (Magenta).

-ve control 1 : ab325555 at a 1/500 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab25631 at a 1/200 dilution followed by ab150081 at a 1/1000 dilution.

• WB

Lab

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 (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 (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/50000 dilution

Predicted band size: 45 kDa

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

false

Exposure time: 20min

• WB

AbReview16707****

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

Predicted band size: 45 kDa

Observed band size: 110 kDa

true

Exposure time: 30s

This image was kindly supplied by Daniel Rodriguez by Abreview

• WB

AbReview42794****

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)

Predicted band size: 45 kDa

Observed band size: 110 kDa

false

Exposure time: 1s

This image is courtesy of an anonymous Abreview

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2 antibody [H4B4] - Lysosome Marker (AB25631)

Immunocytochemistry-immunofluorescence using Anti-LAMP2 antibody [H4B4] - Lysosome Marker, ab25631. Publication image from Solier, S. et al., 2023, Nature, 37100912. Legend direct from paper.

CD44 mediates the uptake of metals in inflammatory macrophages.a, ICP-MS of cellular metals in aMDM treated with an anti-CD44 antibody RG7356 during activation (n = 7 donors). b, ICP-MS of cellular metals in aMDM supplemented with HA (0.6-1 MDa) or permethylated (meth-) HA during activation (n = 6 donors). c, Western blots of hyaluronan synthases (HAS) (n = 6 donors) and ATP7A/B (n = 8 donors) in MDM. d, Fluorescence microscopy of a lysosomal copper(II) probe (Lys-Cu) in aMDM under CD44 knockdown conditions (n = 5 donors). e, Fluorescence microscopy of CTR2 and Lamp2 in aMDM (n = 4 donors). f, Fluorescence microscopy of a lysosomal copper(II) probe (Lys-Cu) in aMDM under CTR2 knockdown conditions (n = 5 donors). For d–f, Scale bars, 10 µm. For a, c, d and f two-sided Mann-Whitney test. For b, Kruskal-Wallis test with Dunn’s post-test. Box plots : boxes represent interquartile range and median, and whiskers indicate the minimum and maximum values. Each colored dot represents a distinct donor for a given panel.Source data