Recombinant Anti-LAMP2 antibody [EPR19531] (ab199947)

All lanes : Anti-LAMP2 antibody [EPR19531] (ab199947) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell
Lane 2 : LAMP2 CRISPR-Cas9 edited HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

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

False colour image of Western blot: Anti-LAMP2 antibody [EPR19531] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab199947 was shown to bind specifically to LAMP2. A band was observed at 100 kDa in wild-type HeLa cell lysates with no signal observed at this size in LAMP2 CRISPR-Cas9 edited cell line ab255402 (CRISPR-Cas9 edited cell lysate ab263861). The band observed in the CRISPR-Cas9 edited lysate lane below 100 kDa is likely to represent a truncated form of LAMP2. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and LAMP2 CRISPR-Cas9 edited HeLa 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-LAMP2 antibody [EPR19531] (ab199947) at 1/1000 dilution

Lane 1 : Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : LAMP2 knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : THP-1 (Human monocytic leukemia cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 45 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab199947 observed at 45 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab199947 was shown to specifically react with LAMP2 in wild-type HEK-293 cells as signal was lost in LAMP2 knockout cells. Wild-type and LAMP2 knockout samples were subjected to SDS-PAGE. Ab199947 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-LAMP2 antibody [EPR19531] (ab199947) at 1/1000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : Human fetal spleen lysate
Lane 3 : Human placenta lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 110-130 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight  observed is consistent with what has been described in the literature (PMID: 19828315) and lots of other products.

All lanes : Anti-LAMP2 antibody [EPR19531] (ab199947) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : THP-1 (Human monocytic leukemia cell line) whole cell lysate
Lane 3 : JAR (Human placenta choriocarcinoma cell line) whole cell lysate
Lane 4 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 45 kDa
Observed band size: 110-130 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight  observed is consistent with what has been described in the literature (PMID: 19828315) and lots of other products.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling LAMP2 with ab199947 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on Human liver is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling LAMP2 with ab199947 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasm staining on Human breast cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.