Recombinant Anti-CD10 antibody [EPR22867-118] (ab256494)

All lanes : Anti-CD10 antibody [EPR22867-118] (ab256494) at 1/1000 dilution

Lane 1 : Rat lung tissue lysate at 10 µg
Lane 2 : Rat kidney tissue lysate at 10 µg
Lane 3 : Human tonsil tissue lysate at 10 µg
Lane 4 : Mouse lung tissue lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

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

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

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

Exposure time: Lane 1/3: 8 seconds Lane 2: 1 second Lane 4: 3 seconds

All lanes : Anti-CD10 antibody [EPR22867-118] (ab256494) at 1/1000 dilution

Lane 1 : Wild type HAP1 whole cell lysate
Lane 2 : CD10 knockout HAP1 whole cell lysate
Lane 3 : Raji (human Burkitts lymphoma B lymphocyte), whole cell lysate
Lane 4 : Ramos (human Burkitts lymphoma B lymphocyte), whole cell lysate
Lane 5 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
Lanes 3-5 : 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?

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

ab256494 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. ab256494 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 molecular weight observed is consistent with what has been described in the literature (PMID:15286660)   Negative control: HT-29 (PMID:19828468).

Exposure time: 59 seconds

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Rat kidney tissue labeling CD10 with ab256494 at 1/100 (5.45 µg/ml) dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution.

Immunohistochemical analysis of 4% PFA fixed 0.2% Triton X-100 permeabilized frozen Mouse kidney tissue labeling CD10 with ab256494 at 1:100 (5.45 µg/ml) dilution followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 (2 µg/ml) dilution. The nuclear counterstain was DAPI (Blue). Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody was ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1:1000 (2 µg/ml) dilution.

CD10 was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab256494 at 1/30 (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab256494 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate 10ug

Lane 2: ab256494 IP in Raji whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab256494 in Raji whole cell lysate

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

Exposure time: 3 min

 

Immunofluorescent analysis of 100% Methanol-fixed 2.4G2 (rat B cell lymphoma B lymphocyte) cells labelling CD10 with ab256494 at 1/500 dilution, followed by Ab256494 anti- CD10 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing staining in 2.4G2 cell line is observed. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is Ab256494 anti- CD10 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunofluorescent analysis of 100% Methanol-fixed WEHI-231 (mouse B cell lymphoma B lymphocyte) cells labelling CD10 with ab256494 at 1/500 dilution, followed by Ab256494 anti- CD10 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing staining in WEHI-231 cell line is observed. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is Ab256494 anti- CD10 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunofluorescent analysis of 100% Methanol-fixed, Ramos (human Burkitt's lymphoma B lymphocyte) cells labelling CD10 with ab256494 at 1/500 dilution, followed by Ab256494 anti- CD10 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green).Confocal image showing membranous staining in Ramos cell line is observed. Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue). Negative control: HT-29 （PMID: 19828468）

Secondary antibody only control: Secondary antibody is Ab256494 anti- CD10 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling CD10 with ab256494 at 1/500 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on renal tubules and of rat kidney (PMID:10705818) is observed. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling CD10 with ab256494 at 1/500 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on renal tubules and of mouse kidney (PMID:10705818) is observed. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling CD10 with ab256494 at 1/500 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on syncytiotrophoblast layer of human placenta (PMID:11092533) is observed. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling CD10 with ab256494 at 1/500 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining on proximal convoluted tubules and glomerular epithelial cells of human kidney (PMID:10705818) is observed. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

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