All lanes : Anti-CD10 antibody [EPR22865-73] (ab255609) at 1/1000 dilution

Lane 1 : Human placenta tissue lysate
Lane 2 : Raji (human Burkitt's lymphoma cell line) whole cell lysate
Lane 3 : Ramos (human Burkitt's lymphoma cell line) whole cell lysate
Lane 4 : HT-29 (human colorectal adenocarcinoma cell line) whole cell lysate
Lane 5 : Wild-type HAP1 whole cell lysate
Lane 6 : CD10 knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 2-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution


Predicted band size: 85 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

ab255609 was shown to specifically react with CD10 in wild-type HAP1 cells as signal was lost in CD10 knockout cells. Wild-type and CD10 knockout samples were subjected to SDS-PAGE. ab255609 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument using the ECL technique.

Lanes 5-6 in this blot were developed using a higher sensitivity ECL substrate.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure times.

Lane 1: 10 seconds; Lanes 2-4: 15 seconds; Lanes 5-6: 70 seconds.

The molecular weight observed is consistent with what has been described in the literature (PMID:15286660).

Negative control: HT-29 (PMID:19828468).

 

CD10 was immunoprecipitated from 0.35 mg of Raji (human Burkitt's lymphoma cell line) whole cell lysate with ab255609 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab255609 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: Raji whole cell lysate 10 μg (Input).
Lane 2: ab255609 IP in Raji whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255609 in Raji whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.

Immunofluorescent analysis of 100% methanol-fixed Ramos (human Burkitt's lymphoma cell line) cells labeling CD10 with ab255609 at 1/50 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 membranous staining in Ramos cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

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.

100% methanol was preferred as fixative.

Immunofluorescent analysis of 100% methanol-fixed Raji (human Burkitt's lymphoma cell line) cells labeling CD10 with ab255609 at 1/50 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 membranous staining in Raji cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

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.

100% methanol was preferred as fixative.

Anti-CD10 antibody [EPR22865-73] (ab255609) at 1/1000 dilution + Mouse kidney tissue lysate at 20 µg

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

Predicted band size: 85 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:15286660).

 

All lanes : Anti-CD10 antibody [EPR22865-73] (ab255609) at 1/1000 dilution

Lane 1 : Rat kidney tissue lysate
Lane 2 : Rat liver tissue 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: 85 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:15286660).

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD10 with ab255609 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on germinal center of human tonsil (PMID:10843287) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a 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 human liver tissue labeling CD10 with ab255609 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on intrahepatic canaliculi of human liver (PMID:10705818) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a 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 human breast tissue labeling CD10 with ab255609 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) ready to use. Membranous staining on myoepithelial cells of human breast (PMID:10705818, 17143263) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a 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 human kidney tissue labeling CD10 with ab255609 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG H&L (HRP) ready to use. Positive staining on proximal convoluted tubules and glomerular epithelial cells of human kidney (PMID:10705818) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a Goat Anti-Rabbit IgG H&L (HRP) ready to use.

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