Overview

  • Product name
    Anti-LAMP2 antibody [H4B4]
    See all LAMP2 primary antibodies
  • Description
    Mouse monoclonal [H4B4] to LAMP2
  • Host species
    Mouse
  • Specificity
    Human CD107b/LAMP-2
  • Tested applications
    Suitable for: Flow Cyt, IHC-Fr, ICC, WB, IHC-P, ICC/IF, IHC-FoFrmore details
  • Species reactivity
    Reacts with: Human, Rhesus monkey, African green monkey
    Does not react with: Mouse, Rat
  • Immunogen

    The details of the immunogen for this antibody are not available.

Properties

Applications

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.

Application Abreviews Notes
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.
ICC/IF 1/100.
IHC-FoFr Use at an assay dependent concentration. (PMID 19837699).

Target

  • Function
    Implicated in tumor cell metastasis. May function in protection of the lysosomal membrane from autodigestion, maintenance of the acidic environment of the lysosome, adhesion when expressed on the cell surface (plasma membrane), and inter-and intracellular signal transduction. Protects cells from the toxic effects of methylating mutagens.
  • Tissue specificity
    Isoform LAMP-2A is highly expressed in placenta, lung and liver, less in kidney and pancreas, low in brain and skeletal muscle. Isoform LAMP-2B is highly expressed in skeletal muscle, less in brain, placenta, lung, kidney and pancreas, very low in liver.
  • Involvement in disease
    Defects in LAMP2 are the cause of Danon disease (DAND) [MIM:300257]; also known as glycogen storage disease type 2B (GSD2B). DAND is a lysosomal glycogen storage disease characterized by the clinical triad of cardiomyopathy, vacuolar myopathy and mental retardation. It is often associated with an accumulation of glycogen in muscle and lysosomes.
  • Sequence similarities
    Belongs to the LAMP family.
  • Post-translational
    modifications
    O- and N-glycosylated; some of the 16 N-linked glycans are polylactosaminoglycans.
  • Cellular localization
    Cell membrane. Endosome membrane. Lysosome membrane. This protein shuttles between lysosomes, endosomes, and the plasma membrane.
  • Information by UniProt
  • Database links
  • Form
    Alternative splicing produces 3 isoforms.
  • Alternative names
    • CD107 antigen like family member B antibody
    • CD107 antigen-like family member B antibody
    • CD107b antibody
    • LAMP 2 antibody
    • LAMP 2C antibody
    • LAMP-2 antibody
    • LAMP2 antibody
    • LAMP2_HUMAN antibody
    • LAMPB antibody
    • LGP 96 antibody
    • LGP110 antibody
    • LGP96 antibody
    • Lysosomal associated membrane protein 2 antibody
    • Lysosome associated membrane glycoprotein 2 antibody
    • Lysosome associated membrane protein 2 antibody
    • Lysosome-associated membrane glycoprotein 2 antibody
    • Lysosome-associated membrane protein 2 antibody
    • MAC3 antibody
    see all

Images

  • 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.

    See Abreview

  • All lanes : Anti-LAMP2 antibody [H4B4] (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.

    See Abreview

  • All lanes : Anti-LAMP2 antibody [H4B4] (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 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.

    See Abreview

  • Anti-LAMP2 antibody [H4B4] (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.

    See Abreview

  • 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.

    See Abreview

  • 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.

  • 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.

References

This product has been referenced in:
  • Johnstone DL  et al. PLPHP deficiency: clinical, genetic, biochemical, and mechanistic insights. Brain 142:542-559 (2019). Read more (PubMed: 30668673) »
  • Liu Y  et al. Prevalence and clinical characteristics of Danon disease among patients with left ventricular hypertrophy and concomitant electrocardiographic preexcitation. Mol Genet Genomic Med 7:e638 (2019). Read more (PubMed: 30929317) »
See all 106 Publications for this product

Customer reviews and Q&As

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1-10 of 16 Abreviews

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (neuroglioma cells)
Permeabilization
Yes - Saponin/Glycine
Specification
neuroglioma cells
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted May 01 2019

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa Cells)
Permeabilization
Yes - 0.3% Triton X-100
Specification
HeLa Cells
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Nov 01 2018

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing (12.5)
Sample
Rhesus monkey Cell lysate - whole cell (Rhesus monkey primary Retinal pigmented epithelium)
Specification
Rhesus monkey primary Retinal pigmented epithelium
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: RT°C

Abcam user community

Verified customer

Submitted May 13 2014

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Skeletal muscle from the gastrocnemius)
Specification
Skeletal muscle from the gastrocnemius
Fixative
Methacarn
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris pH 9.0
Permeabilization
No
Blocking step
Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Dec 20 2012

Application
Western blot
Sample
Human Tissue lysate - other (Skeletal Muscle)
Loading amount
20 µg
Specification
Skeletal Muscle
Gel Running Conditions
Reduced Denaturing (12% Bis-Tris gel)
Blocking step
Li-Cor Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Dec 20 2012

Application
Immunocytochemistry/ Immunofluorescence
Sample
Monkey Cell (FRhK-4 cells)
Specification
FRhK-4 cells
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.2%Triton-X100 in PBS
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Nov 07 2012

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Huh7)
Specification
Huh7
Fixative
Paraformaldehyde
Permeabilization
Yes - saponin
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 21 2012

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Human Fetal Lung Fibroblasts)
Specification
Human Fetal Lung Fibroblasts
Fixative
Formaldehyde
Permeabilization
Yes - Triton-X 0.3%
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 17 2011

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (human melanoma MNT-1)
Specification
human melanoma MNT-1
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% triton/PBS
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Mar 23 2011

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Hela cell)
Specification
Hela cell
Fixative
Formaldehyde
Permeabilization
Yes - triton
Blocking step
2%BSA and2%Goat serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jul 26 2010

1-10 of 16 Abreviews

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