Anti-LAMP1 抗体 [EPR24395-31] (ab278043)

All lanes : Anti-LAMP1 antibody [EPR24395-31] (ab278043) at 1/1000 dilution

Lane 1 : Wild-type HCT 116 cell lysate
Lane 2 : LAMP1 knockout HCT 116 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 100-120 kDa why is the actual band size different from the predicted?

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 : Anti-LAMP1 antibody [EPR24395-31] (ab278043) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 45 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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

 

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

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.

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. 

 

 

All lanes : Anti-LAMP1 antibody [EPR24395-31] (ab278043) at 1/1000 dilution

Lane 1 : Human kidney tissue lysate
Lane 2 : Human spleen tissue lysate
Lane 3 : Human colon cancer tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution (VeriBlot for IP secondary antibody(HRP))

Predicted band size: 45 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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 : Anti-LAMP1 antibody [EPR24395-31] (ab278043) at 1/1000 dilution

Lane 1 : Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell) whole cell lysate
Lane 2 : LAMP1 knockout HAP1 whole cell lysate
Lane 3 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : Human kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at 1/10000 dilution

Predicted band size: 45 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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.

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.

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

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

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized LAMP1 KO HAP1 cells labelling LAMP1 with ab278043 at 1/50 (10.9 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.

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.