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

Lane 1 : Wild-type HEK-293 cell lysate
Lane 2 : LAMP2 knockout HEK-293 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : LAMP2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 45 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab199947 observed at 110 kDa. Red - loading control, ab8245 observed at 37 kDa.

 ab199947 was shown to react with LAMP2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255402 (knockout cell lysate ab263861) was used. Wild-type and LAMP2 knockout samples were subjected to SDS-PAGE. ab199947 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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 20000 dilution for 1 hour at room temperature before imaging.

 

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

Lane 1 : Wild-type HEK-293 whole cell lysate
Lane 2 : LAMP2 knockout HEK-293 whole cell lysate
Lane 3 : HepG2 whole cell lysate
Lane 4 : THP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

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.