All lanes : Anti-S100A4 antibody [EPR14639(2)] (ab197896) 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: 12 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab197896 observed at 12 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab197896 Recombinant Anti-S100A4 antibody [EPR14639(2)] was shown to specifically react with S100A4 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261758 (knockout cell lysate ab257045) was used. Wild-type and S100A4 knockout samples were subjected to SDS-PAGE. ab197896 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) were incubated overnight at 4°C at 1 in 1000 dilution 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.

 

All lanes : Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1 : Wild-type A549 whole cell lysate
Lane 2 : S100A4 knockout A549 whole cell lysate
Lane 3 : A375 whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Lanes 1 - 4: Merged signal (red and green). Green - ab197896 observed at 12 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab197896 was shown to recognize S100A4 in wild-type A549 cells as signal was lost at the expected MW in S100A4 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and S100A4 knockout samples were subjected to SDS-PAGE. Ab197896 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling S100A4 with ab197896 at 1/2000 dilution (0.376 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on lymphocytes of rat spleen.The section was incubated with ab197896 for 30 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

All lanes : Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lanes 1 & 5 : A549 whole cell lysate
Lane 2 : Human liver lysates
Lane 3 : Human lung lysates
Lane 4 : Human colon lysates

Lysates/proteins at 20 µg per lane.

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

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


Exposure time: 26 seconds

Immunohistochemical analysis of paraffin-embedded Human gastric carcinoma tissue labeling S100A4 using ab197896 at 1/2000 dilution. Goat Anti-Rabbit IgG H&L (HRP) ab97051 was used as a secondary antibody at 1/500 dilution. Cells were counterstained with Hematoxylin. 

Inset image: negative control obtained using PBS instead of ab197896 and secondary antibody only.
Note: Cytoplasm and nuclear staining on human gastric carcinoma tissue was observed.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1 : A375 (human malignant melanoma) whole cell lysate
Lane 2 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 12 kDa


Exposure time: 3 minutes

Blocking/dilution buffer: 5% NFDM/TBST

Flow Cytometry analysis of Jurkat cells labelling S100A4 with ab197896 at 1/250 (red). Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

ab197896 at 1/40 immunoprecipitating S100A4 in A549 whole cell lysate observed at 12 KDa.

Lane 1 (input): A549 whole cell lysate 10μg

Lane 2 (+): ab197896 + A549 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab197896 in A549 whole cell lysate

For western blotting, Panel A: ab197896, 1:1000; Panel B: ab124805, 1:1000 and anti-rabbit IgG (HRP), specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

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

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling S100A4 using ab197896 at 1/2000 dilution. Goat Anti-Rabbit IgG H&L (HRP) ab97051 was used ay 1/500 dilution as a secondary antibody and cells were counterstained with Hematoxylin.

Inset image: negative control obtained using PBS instead of ab197896 and secondary antibody only.
Note: Nuclear and cytoplasm staining on cervix carcinoma tissue was observed.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labeling S100A4 using ab197896 at 1/2000 dilution. Goat Anti-Rabbit IgG H&L (HRP) ab97051 was used as a secondary antibody at a dilution of 1/500 and cells were counterstained with Hematoxylin.

Inset image: negative control obtained using PBS instead of ab197896 and secondary antibody only.
Note: Nuclear and weakly staining on lung carcinoma tissue was observed.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1 : A549 (Human lung carcinoma epithelial cell) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 3 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates
Lane 4 : Mouse spleen tissue lysates
Lane 5 : Mouse bone marrow tissue lysates
Lane 6 : Mouse heart tissue lysates

Lysates/proteins at 20 µg per lane.

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

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


Exposure time: 44 seconds

Blocking and dilution buffer: 5% NFDM/TBST.

Some optimisation may be required for detection of the target protein due to low levels of endogenous expression in some samples.