Mouse, Human, Monkey, Chinese hamster Predicted to work with:
Synthetic peptide corresponding to Human LAMP2A aa 350 to the C-terminus (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH). LAMP2 has 3 distinct isoforms, LAMP2A, 2B & 2C. Database link: P13473 (Peptide available as ab23322)
This antibody gave a positive signal in Western Blot in MEF1 whole cell lysate and the following tissue lysates: Mouse Lung, Human Liver, Human Liver (membrane fraction).
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 0.5 - 1 µg/ml. Detects a band of approximately 105 kDa (predicted molecular weight: 100 kDa).
Abcam recommends using 3% milk as the blocking agent.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration.
ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
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.
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
Belongs to the LAMP family.
O- and N-glycosylated; some of the 16 N-linked glycans are polylactosaminoglycans.
Cell membrane. Endosome membrane. Lysosome membrane. This protein shuttles between lysosomes, endosomes, and the plasma membrane.
ab18528 stained MEF1 cells. The cells were 4% formaldehyde fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18528 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - Anti-LAMP2A antibody (ab18528)This image and legend is taken from an Abreview by Eeva-Liisa Eskelinen submitted in December 2007
Lanes 1-3 : Anti-LAMP2A antibody (ab18528) at 1 µg/ml Lanes 4-5 : non Abcam anti-LAMP2 antibody
Lanes 1 & 5 : MEF lysate extracted using 0.5 % NP40 Lane 2 : HeLa lysate extracted using 1%Triton X-100 Lane 3 : HeLa lysate extracted using 0.5 % NP40 Lane 4 : MEF lysate extracted using 1% Triton X-100
Secondary All lanes : goat anti-rabbit at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 100 kDa
Western blotting with the anti-LAMP2A (lanes 1-3) and an alternative antibody against the luminal domain of mouse LAMP2 (lanes 4 & 5) using MEF & HeLa cells extracted using either 1% Triton X-100 or 0.5 % NP40. Ab18528 recognized a 100 kDa band corresponding intact LAMP2 and also recognized mouse LAMP2 as expected from the sequence similarity. LAMP2 undergoes extensive glycosylation so ab18528 detects a band of ~ 100 kDa by Western blot.
IHC image of Lamp2A staining in human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab18528, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ab18528 staining Lamp2A in CaCO2 cells treated with SB 202190 (ab120638), by ICC/IF. Increase of Lamp2A expression correlates with increased concentration of SB 202190, as described in literature. The cells were incubated at 37°C for 3 hours in media containing different concentrations of ab120638 (SB 202190) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab18528 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
Western blot - Anti-LAMP2A antibody (ab18528)
All lanes : Anti-LAMP2A antibody (ab18528) at 1 µg/ml (blocked with 3% Milk)
Lane 1 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate Lane 2 : Lung (Mouse) Tissue Lysate Lane 3 : Human liver tissue lysate - total protein (ab29889) Lane 4 : Human liver left lobe tissue lysate - membrane extract (ab29086)
Lysates/proteins at 20 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
ICC/IF image of ab18528 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18528, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
ICC/IF image of ab18528 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18528, 5µg/ml) overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Magalhaes J et al. Effects of ambroxol on the autophagy-lysosome pathway and mitochondria in primary cortical neurons. Sci Rep8:1385 (2018).
Read more (PubMed: 29362387) »
Zhang S et al. Sorting nexin 10 acts as a tumor suppressor in tumorigenesis and progression of colorectal cancer through regulating chaperone mediated autophagy degradation of p21Cip1/WAF1. Cancer Lett419:116-127 (2018).
Read more (PubMed: 29355659) »