All lanes : Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/2000 dilution (purified)

Lane 1 : Mouse kidney tissue lysate
Lane 2 : Rat kidney tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

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

Blocking and dilution buffer: 5% NFDM/TBST.

Difference in MW may be caused by different degree of glycosylation.

Immunohistochemical analysis of paraffin-embedded Human placenta labeling LAMP2A with unpurified ab125068 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic and membrane staining on human placenta tissue 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 before commencing with IHC staining protocol.

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling LAMP2A (red) with ab125068 at a 1/1000 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

ab125068 (purified) at 1/60 immunoprecipitating LAMP2A in HeLa whole cell lysate.

Lane 1 (input): HeLa whole cell lysate (10µg)

Lane 2 (+): ab125068 + HeLa whole cell lysate (10µg).

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab125068 in HeLa whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1500 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/10000 dilution (purified)

Lane 1 : Jurkat cell lysate
Lane 2 : ECV-304 cell lysate
Lane 3 : JAR cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

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

Blocking and dilution buffer: 5% NFDM/TBST.

May be seen at ~50 kDa representing the unglycosylated isoforms of LAMP2 and ~120 kDa representing the glycosylated form.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue labelling LAMP2A with purified ab125068 at 1/200. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

LAMP2A was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with unpurified ab125068 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab125068 at 1/2000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.

Lane 1: HeLa whole cell lysate 10ug (Input).

Lane 2: ab125068 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab125068 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

 

Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/5000 dilution (unpurified) + HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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


Exposure time: 3 minutes

Blocking and Diluting buffer and concentration: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded Human liver labeling LAMP2A with unpurified ab125068 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic and membrane staining on human liver tissue 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 before commencing with IHC staining protocol.

LAMP2A was immunoprecipitated from 1mg of RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate with ab125068 at 1/100 dilution. Western blot was performed from the immunoprecipitate using unpurified ab125068 at 1/2000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.

Lane 1: RAW 264.7 whole cell lysate 10ug (Input).

Lane 2: ab125068 IP in RAW 264.7 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab125068 in RAW 264.7 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

All lanes : Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/5000 dilution (unpurified)

Lane 1 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 2 : ECV-304 (Human urinary bladder cancer cell line) whole cell lysate
Lane 3 : JAR (Human placenta choriocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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


Exposure time: 30 seconds

Blocking and Diluting buffer and concentration: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded Mouse liver labeling LAMP2A with unpurified ab125068 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic and membrane staining on mouse liver tissue 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 before commencing with IHC staining protocol.

All lanes : Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/2000 dilution (unpurified)

Lane 1 : Mouse kidney
Lane 2 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

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


Exposure time: 3 minutes

Blocking and Diluting buffer and concentration: 5% NFDM /TBST

Immunohistochemical analysis of paraffin-embedded Rat liver labeling LAMP2A with unpurified ab125068 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic and membrane staining on rat liver tissue 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 before commencing with IHC staining protocol.

All lanes : Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/2000 dilution (unpurified)

Lane 1 : Rat liver
Lane 2 : Rat kidney
Lane 3 : C6 (Rat glial tumor cells) whole cell lysate
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 45 kDa


Exposure time: 3 minutes

Blocking and Diluting buffer and concentration: 5% NFDM/TBST.