Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

Side-by-side comparison of ICC performance using the rabbit polyclonal ab18528 and RabMab® ab125068. Staining was performed on wild-type HeLa cells (top panel) and LAMP2 knockout HeLa cells (bottom panel, available as ab255402). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Tween-20 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab18528 or ab125068 overnight at +4°C at 3 different concentrations : 1.0 μg/mL, 0.2 μg/mL and 0.04 μg/mL. Secondary antibody incubation was at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) (shown in green) at 1/1000 and nuclear DNA was labelled with DAPI (shown in blue). Images were acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Some cytoplasmic cross-reactivity is seen using ab18528 at 1.0 μg/mL, but further titration of the antibody improves the ICC staining result. The RabMab® ab125068 shows negligible non-specific staining across the dilution range. Quantification of the antibody signal was performed using a minimum of 180 cells and data are presented as mean ± SD.Optimal dilutions/concentrations may vary across different cell types/experiment conditions and should be determined by the end user.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

ab125068 staining LAMP2a in wild-type HeLa cells (top panel) and LAMP2 knockout HeLa cells (bottom panel, available as ab255402). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Tween-20 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab125068 at 0.04 μg/mL and ab7291 at 1 μg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) (shown in green) and goat secondary antibody to Mouse IgG (Alexa Fluor^® 594) (ab150120) (shown in red) both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue).

In our hands, permeabilization with 0.1% Triton X-100 (5 min) resulted in greatly reduced signal and we recommend using 0.1% Tween-20 (5 min) for detecting this target.

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

ab125068 staining LAMP2a in wild-type HeLa cells (top panel) and LAMP2 knockout HeLa cells (bottom panel, available as ab255402). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Tween-20 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab125068 at 0.04 µg/mL and ab7291 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) (shown in green) and goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120) (shown in red) both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). In our hands, permeabilization with 0.1% Triton X-100 (5 min) resulted in greatly reduced signal and we recommend using 0.1% Tween-20 (5 min) for detecting this target. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

Intracellular 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 Fluorr^® 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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

Side-by-side comparison of ICC performance using the rabbit polyclonal ab18528 and RabMab® ab125068. Staining was performed on wild-type HeLa cells (top panel) and LAMP2 knockout HeLa cells (bottom panel, available as ab255402). The cells were fixed with 4% PFA (10 min), permeabilized with 0.1% Tween-20 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab18528 or ab125068 overnight at +4°C at 3 different concentrations : 1.0 μg/mL, 0.2 μg/mL and 0.04 μg/mL. Secondary antibody incubation was at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) (shown in green) at 1/1000 and nuclear DNA was labelled with DAPI (shown in blue). Images were acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown. Some cytoplasmic cross-reactivity is seen using ab18528 at 1.0 μg/mL, but further titration of the antibody improves the ICC staining result. The RabMab® ab125068 shows negligiblenon-specific staining across the dilution range. Quantification of the antibody signal was performed using a minimum of 135 cells and data are presented as mean ± SD.Optimal dilutions/concentrations may vary across different cell types/experiment conditions and should be determined by the end user.

• IP

Lab

Immunoprecipitation - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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:

Immunoprecipitation - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068)

Predicted band size: 45 kDa

Observed band size: 120 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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.

All lanes:

Immunoprecipitation - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068)

Predicted band size: 45 kDa

Observed band size: 120 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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:

Immunoprecipitation - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068)

Predicted band size: 45 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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.

All lanes:

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/10000 dilution

Lane 1:

Jurkat cell lysate at 20 µg

Lane 2:

ECV-304 cell lysate at 20 µg

Lane 3:

JAR cell lysate at 20 µg

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

false

• WB

Supplier Data

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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

All lanes:

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/5000 dilution

Lane 1:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 20 µg

Lane 2:

ECV-304 (Human urinary bladder cancer cell line) whole cell lysate at 20 µg

Lane 3:

JAR (Human placenta choriocarcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 37 kDa,45 kDa

Observed band size: 120 kDa

false

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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

All lanes:

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/5000 dilution

All lanes:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg

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

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

False colour image of Western blot : Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab125068 was shown to bind specifically to LAMP2A. 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 LAMP2A. 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:

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/2000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

LAMP2 CRISPR-Cas9 edited HeLa cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

Blocking and diluting buffer and concentration : 5% NFDM /TBST. ab181602 was used as a GAPDH loading control. LAMP2A is highly expressed in placenta, lung and liver, less in kidney and pancreas, low in brain and skeletal muscle (PMID : 10212251PubMed : 7488019, PubMed : 26856698). For better using it in tissue with low expression level, we suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate).

All lanes:

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/1000 dilution

All lanes:

Western blot 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 1/20000 dilution

Predicted band size: 45 kDa

Observed band size: 100 kDa

false

Exposure time: 40s

• WB

Supplier Data

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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

All lanes:

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/2000 dilution

Lane 1:

Mouse kidney at 10 µg

Lane 2:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 3:

NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

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

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

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

All lanes:

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/2000 dilution

Lane 1:

Rat liver at 10 µg

Lane 2:

Rat kidney at 10 µg

Lane 3:

C6 (Rat glial tumor cells) whole cell lysate at 10 µg

Lane 4:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 45 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (AB125068)

Blocking and dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-LAMP2A antibody [EPR4207(2)] - Lysosome Marker (ab125068) at 1/2000 dilution

Lane 1:

Mouse kidney tissue lysate at 20 µg

Lane 2:

Rat kidney tissue lysate at 20 µg

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

false