All lanes : Anti-Acid phosphatase antibody [EPR21791] (ab235449) at 1/1000 dilution

Lane 1 : Wild-type HEK-293 cell lysate
Lane 2 : ACP1 knockout HEK-293 cell lysate
Lane 3 : K562 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 18 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab235449 observed at 18 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab235449 was shown to react with Acid phosphatase in wild-type HEK-293 cells in western blot with loss of signal observed in ACP1 knockout sample. Wild-type and ACP1 knockout HEK-293 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab235449 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling Acid Phosphatase with ab235449 at 1/4000 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 cervix adenocarcinoma epithelial cell) cells labeling Acid Phosphatase with ab235449 at 1/100 dilution, followed by ab150077 Alexa-Fluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Confocal image showing cytoplasmic and nuclear staining in HeLa cell line (PMID 26159288) is observed. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as the counterstain (Red). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa-Fluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

All lanes : Anti-Acid phosphatase antibody [EPR21791] (ab235449) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : HepG2 (human hepatocellular carcinoma epithelial cell), whole cell lysate
Lane 3 : HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 4 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate
Lane 5 : Human placenta tissue lysate

Lysates/proteins at 20 µg per lane.

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

Blocking/Diluting buffer and concentration: 5% NFDM/TBST
Exposure time: 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 cervix adenocarcinoma epithelial cell) cell line labeling Acid Phosphatase with ab235449 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control, and an unlabeled control (Cells without incubation with primary antibody and secondary antibody, Blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HepG2 (Human hepatocellular carcinoma epithelial cell) cells labeling Acid Phosphatase with ab235449 at 1/100 dilution, followed by ab150077 Alexa-Fluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution (Green). Confocal image showing cytoplasmic and nuclear staining in HepG2 cell line (PMID 26159288) is observed. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used as the counterstain (Red). The nuclear counterstain is DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Alexa-Fluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human prostatic hyperplasia tissue labeling Acid Phosphatase with ab235449 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining in human prostatic hyperplasia (PMID: 26159288) 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).