Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker

• mIHC

Supplier Data

Multiplex immunohistochemistry - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human thyroid tissue staining FUCA1 with ab323794 at a 1 : 1000 (0.511 ug/ml) dilution, ab278043 anti-LAMP1 used at 1 : 5000 (0.103 ug/ml) dilution.

Panel A : merged staining of anti-FUCA1 (green; Opal™520), anti-LAMP1 (magenta; Opal™570) on human thyroid.
Panel B : anti-FUCA1 showed granular staining in human thyroid.
Panel C : ant-LAMP1 staining lysosome in human thyroid.
Nuclear DNA was labeled with DAPI (shown in blue).

The section was incubated in three rounds of staining : in the order of ab323794 and ab278043 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

Nuclear counter stain with DAPI.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Intracellular flow cytometric analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labelling LAMP1 with ab278043 at 1/500 dilution (red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Intracellular flow cytometric analysis of Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell, Right)/ LAMP1 knockout HAP1 cells (Left) cells labelling LAMP1 with ab278043 at 1/500 dilution (red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized LAMP1 KO HAP1 cells labelling LAMP1 with ab278043 at 1/100 (5.45 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 2 ug/ml dilution (green). Confocal image showing cytoplasmic staining in parental HAP1 cell line and no staining in LAMP1 KO HAP1 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody- Microtubule Marker (Alexa Fluor ^® 594) was used to counterstain at 1/200 (red).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling LAMP1 with ab278043 at 1/100 (5.45 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (green). Confocal image showing cytoplasmic staining in HeLa. ab25630 Anti-LAMP1 mouse monoclonal antibody was used to counterstain at 1/500 (red). The Nuclear counterstain was DAPI (blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Immunohistochemical analysis of paraffin-embedded Human lung cancer tissue labelling LAMP1 with ab278043 at 1/5000 (0.109 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) was used. Cytoplasmic staining in human lung cancer. The section was incubated with ab278043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling LAMP1 with ab278043 at 1/5000 (0.109 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) was used. Cytoplasmic staining in human liver. The section was incubated with ab278043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labelling LAMP1 with ab278043 at 1/5000 (0.109 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) was used. Cytoplasmic staining in human pancreas. The section was incubated with ab278043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Immunohistochemical analysis of paraffin-embedded Human tonsil labelling LAMP1 with ab278043 at 1/5000 (0.109 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) was used. Cytoplasmic staining in human tonsil. The section was incubated with ab278043 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond® Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• WB

Lab

Western blot - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Blocking buffer and Diluting buffer : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

Lanes 1-4 : Merged signal (red and green). Green - ab278043 observed at 110 kDa. Red - loading control ab8245 observed at 36 kDa.

ab278043 Anti-LAMP1 antibody [EPR24395-31A] was shown to specifically react with LAMP1 in wild-type HAP1 cells. Loss of signal was observed when knockout cell line was used. Wild-type and LAMP1 knockout samples were subjected to SDS-PAGE. ab278043 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (ab278043) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate at 20 µg

Lane 2:

LAMP1 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Human kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 45 kDa

Observed band size: 110 kDa

false

• WB

Lab

Western blot - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Blocking and Diluting buffer : 5% NFDM/TBST

LAMP1 is a glycoprotein. The molecular weight observed is consistent with what has been described in the literature (PMID : 27061067, 15111122).

Exposure time : 3 minutes

All lanes:

Western blot - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (ab278043) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/50000 dilution

Predicted band size: 45 kDa

Observed band size: 110 kDa

false

• WB

Lab

Western blot - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Blocking and Diluting buffer : 5% NFDM/TBST

LAMP1 is a glycoprotein. The molecular weight observed is consistent with what has been described in the literature (PMID : 27061067, 15111122).

Exposure time : 3 minutes

All lanes:

Western blot - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (ab278043) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

Human spleen tissue lysate at 20 µg

Lane 3:

Human colon cancer tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 45 kDa

Observed band size: 110 kDa

false

• WB

Lab

Western blot - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (AB278043)

Western blot : Anti-LAMP1 antibody [EPR24395-31] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab278043 was shown to bind specifically to LAMP1. A band was observed at 100-120 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in LAMP1 knockout cell line. To generate this image, wild-type and LAMP1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-LAMP1 antibody [EPR24395-31] - Lysosome Marker (ab278043) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

LAMP1 knockout HCT 116 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Observed band size: 100-120 kDa

false