This is a knockout-validated antibody summary, based on the publication "LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes", as cited below [1]. Labome curates formal publications to compile a list of antibodies with unambiguous specificity within Validated Antibody Database (VAD).

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Figure 1. Western blot analysis of total cell extracts confirm the absence of LAMP-2 expression in single and double knock out while LAMP-1 was not expressed in LAMP-1/2 -/- fibroblasts.

Rat monoclonal IgG2a

Company: Developmental Studies Hybridoma Bank

Antibody: LAMP-2

Catalog number: ABL-93

Summary: Rat monoclonal IgG2a against an epitope corresponding to the luminal region of LAMP-2 from BALB/c 3T3 mouse embryo fibroblast tissue culture cell glycoproteins purified by lectin chromatography. Reacts with mouse protein. Suitable for western blot, immunofluorescence, immunohistochemistry, immunoprecipitation, and FACS.

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Figure 2. Immunofluorescence shows that LAMP-2 is expressed in wild type MEF while LAMP-1 is expressed in wild type and LAMP-2 deficient cells but not in doubly deficient fibroblast. Scale bar = 20µm. From [1].

Western blot | Immunocytochemistry

Wild type, LAMP-2-/- and LAMP-1/2-/- MEFs. WB: resuspended in lysis buffer (125 mM Tris-HCl pH 6.8/4% SDS/20% glycerol/200 mM DTT) and subjected to sonication. IC: cells were washed with PBS and fixed with freshly prepared 4% paraformaldehyde.

WB: for 1 h at room temperature.

IC: 10% goat serum/1% donkey serum for 30 min at room temperature.

WB: overnight at 4°C.

IC: diluted in 1% goat serum/1% donkey serum for 1 h at room temperature.

WB: alkaline phosphatase coupled antibody for 1 h at room temperature.

IC: Alexa Fluor 488 Goat anti-rat IgG G (Invitrogen, A-11006), diluted in goat/donkey serum 1% was added to the cells for 1 h at room temperature

WB: DuoLux chemiluminescent substrate (Vector Laboratories, SK 6604, Burlingame, CA, USA) the signal was analyzed using the Lumi-Imager F1.

IC: confocal laser scanning microscope (Lsm 780, Carl Zeiss) using ZEN 2010 or ZEN 2012 software. This microscope was equipped with 63×1.4NA oil objective lens, a diode laser 405 nm, an argon laser, multiline, 458 nm, 488 nm, 514 nm, a DPSS-Laser 561 nm, a HeNe-laser 594 nm and a HeNe-laser 633 nm

The same clone (ABL-93) is sold as BioLegend 816101; Santa Cruz Biotechnology sc-20004; Abnova MAB3266, MAB5837, MAB5838; Invitrogen RMCD107B01, A15449; LifeSpan Biosciences LS-C196123, LS-C350582, LS-C350586, LS-C350607; Developmental Studies Hybridoma Bank ABL-93; EMD Millipore MABC40.