Recombinant Anti-S100A4 antibody [EPR2761(2)] - BSA and Azide free (ab216003)

All lanes : Anti-S100A4 antibody [EPR2761(2)] (ab124805) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : S100A4 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 12 kDa
Observed band size: 11 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab124805).

Lanes 1 - 2: Merged signal (red and green). Green - ab124805 observed at 11 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab124805 was shown to react with S100A4 in wild-type HeLa cells in Western blot with loss of signal observed in S100A4 knockout cell line ab265709 (S100A4 knockout cell lysate ab257046). Wild-type HeLa and S100A4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab124805 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) 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 h at room temperature before imaging.

All lanes : Anti-S100A4 antibody [EPR2761(2)] (ab124805) at 1/1000 dilution

Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : S100A4 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : A-375 (Human malignant melanoma cell line) whole cell lysate
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 12 kDa
Observed band size: 12 kDa

This data was developed using the same antibody clone in a different buffer formulation (ab124805).

Lanes 1 - 4: Merged signal (red and green). Green - ab124805 observed at 12 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab124805 was shown to react with S100A4 in wild-type A549 cells in Western blot with loss of signal observed in S100A4 knockout sample. Wild-type A549 and S100A4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab124805 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 h at room temperature before imaging.

ab124805 staining S100A4 in human tonsil tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124805).

ab124805 staining S100A4 in HeLa (human cervix adenocarcinoma) by intracellular flow cytometry. Cells were fixed with 80% methanol and permeabilised with 0.1% Triton X-100 (in PBS). The sample was incubated with the primary antibody at a dilution of 1/800. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody. 

Isoytype control: ab172730 rabbit monoclonal IgG (Black) 

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue) 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124805). 

 

 

ab124805 staining S100A4 in HeLa (human cervix adenocarcinoma) by intracellular flow cytometry. Cells were fixed with 80% methanol and permeabilised with 0.1% Triton X-100 (in PBS). The sample was incubated with the primary antibody at a dilution of 1/80. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody. 

Isoytype control: ab172730 rabbit monoclonal IgG (Black) 

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue) 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124805). 

 

 

ab124805 immunoprecipitating S100A4. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/30 and Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at a dilution of 1/1500.

Lane 1: Human tonsil whole cell lysate (10ug)
Lane 2: Human tonsil whole cell lysate 
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab124805 in human tonsil whole cell lysate

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124805).

ab124805 staining S100A4 in Jurkat (human acute T cell leukemia) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at 1/500. ab7291 and ab150120 were used as counterstains for primary antibody ab124805 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124805).

Unpurified ab124805 staining S100A4 in the A549 cell line from Human lungs by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 0.25% in PBS. Samples were incubated with primary antibody (1/100) for 45 minutes at 25°C. A TRITC-conjugated Goat anti-rabbit  polyclonal was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124805).

See Abreview

Unpurified ab124805, at 1/250 dilution, staining S100A4 in paraffin-embedded Human tonsil tissue by Immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124805).

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

Immunohistochemical analysis of Human lung tissue, staining S100A4 with unpurified ab124805.

Tissue was fixed with HOPE and blocked with blocking solution for 5 minutes at 25°C. Samples were incubated with primary antibody (1/1000 in diluent) for 1 hour at 25°C. An undiluted HRP-conjugated goat anti-rabbit polyclonal IgG was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab124805).

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

See Abreview