All lanes : Anti-SP1 antibody [EPR6662(B)] (ab124804) at 1/5000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SP1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Lanes 1-2: Merged signal (red and green). Green - ab124804 observed at 100 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab124804 Anti-SP1 antibody [EPR6662(B)] was shown to specifically react with SP1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265519 (knockout cell lysate ab257698) was used. Wild-type and SP1 knockout samples were subjected to SDS-PAGE. ab124804 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

 

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SP1 with Purified ab124804 at 1:100 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SP1 with Purified ab124804 at 1:200 dilution (4 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with None. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric carcinoma tissue sections labeling SP1 with Purified ab124804 at 1:2000 dilution (0.41 µg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Purified)ImmunoHistoProbe one step HRP Polymer (ready to use) was used for detection. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

All lanes : Anti-SP1 antibody [EPR6662(B)] (ab124804) at 1/5000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 20 µg


Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

We are unsure how to define the extra bands.

Anti-SP1 antibody [EPR6662(B)] (ab124804) at 1/5000 dilution (Purified) + Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

We are unsure how to define the extra bands.

All lanes : Anti-SP1 antibody [EPR6662(B)] (ab124804) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : SP1 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 81 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab124804 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.

Ab16032 detected the expected band for SP1 in wild-type HAP1 cells along with additional cross-reactive bands. The band was not seen in SP1 knockout HAP1 cells. Wild-type and SP1 knockout samples were subjected to SDS-PAGE. ab124804 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

 

 

ab124804, at 1/100 dilution staining SP1 in paraffin-embedded Human colon tissue, by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

All lanes : Anti-SP1 antibody [EPR6662(B)] (ab124804)

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : SP1 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 81 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab124804 observed at 100 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab124804 and a competitor's top cited rabbit polyclonal antibody.

Equilibrium disassociation constant (K_D)
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