All lanes : Anti-Acid phosphatase antibody [EPR21787] (ab235448) at 1/1000 dilution

Lane 1 : Wild-type HEK293 whole cell lysate
Lane 2 : ACP1 knockout HEK293 whole cell lysate
Lane 3 : K562 whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 18 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab235448 observed at 18 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab235448 was shown to specifically react with ACP1 (Acid phosphatase 1) in wild-type HEK 293 cells as signal was lost in ACP1 knockout cells. Wild-type and ACP1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab235448 and ab8245 (Mouse anti GAPDH 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.

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling Acid phosphatase with ab235448 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in human colon cancer (PMID: 25811796) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Acid phosphatase with ab235448 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and nuclear staining in HeLa cell line (PMID 26159288). The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes : Anti-Acid phosphatase antibody [EPR21787] (ab235448) at 1/1000 dilution

Lane 1 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg
Lane 2 : SH-SY5Y (human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg
Lane 3 : HCT 116 (human colorectal carcinoma cell line) whole cell lysate at 20 µg
Lane 4 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg
Lane 5 : Human placenta lysate at 20 µg
Lane 6 : Human colon lysate at 20 µg
Lane 7 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg
Lane 8 : C6 (rat glioma cell line) whole cell lysate at 10 µg
Lane 9 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate at 10 µg

Secondary
Lanes 1-6 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Lanes 7-9 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 18 kDa
Observed band size: 18 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 37 seconds, Lanes 2-9: 3 minutes.

The molecular mass observed is consistent with what has been described in the literature (PMID:25811796).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cell line labeling Acid phosphatase with ab235448 at 1/50 dilution (red) compared with a Isotype control details (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

Acid phosphatase was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab235448 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab235448 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate lysate 10 μg (Input).
Lane 2: ab235448 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab235448 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 3 minutes.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Acid phosphatase with ab235448 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in rat colon (PMID: 25811796) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

 

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Acid phosphatase with ab235448 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in mouse colon (PMID: 25811796) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling Acid phosphatase with ab235448 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic and nuclear staining in HepG2 cell line (PMID 26159288). The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.