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-1

Catalog number: 1DB4

Summary: Rat monoclonal IgG2a against NIH/3T3 mouse embryo fibroblast tissue culture cell membranes. Reacts with mouse by western blot, immunofluorescence, immunohistochemistry, immunoprecipitation, and ELISA.

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Figure 2. 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

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

WB: for 1 h at room temperature.

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

WB: overnight at 4°C.

ICC: diluted in 1% goat serum/1% donkey serum overnight at 4°C or 1 h at room temperature.

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

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

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

ICC: 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.

LAMP-2 (clone ABL-93, Developmental Studies Hybridoma Bank is also KO validated (WB and ICC) in the same Figure.

The same clone (1D4B) is sold as Bio-Rad MCA4707T; BioLegend 121601, 121602, 121603, 121604, 121605, 121606, 121607, 121608, 121609, 121610, 121611, 121612, 121613, 121614, 121615, 121616, 121617, 121619, 121620, 121622, 121623, 121624, 121625, 121626; Santa Cruz Biotechnology sc-19992; LifeSpan Biosciences LS-C8571, LS-C8573, LS-C84918, LS-C189726, LS-C196121, LS-C196122, LS-C350569, LS-C350571, LS-C350572; Abnova MAB3265, MAB5835, MAB5836; Abcam ab25245; Invitrogen RMCD107A01; MyBioSource MBS673717; BD Biosciences 553792, 560647, 560646, 560648, 561069, 553793, 558661, 564348, 564347; Developmental Studies Hybridoma Bank 1D4B; EMD Millipore MABC39.