Anti-S100A4 antibody [EPR14639(2)]

• WB

Lab

Western blot - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

False colour image of Western blot : Anti-S100A4 antibody [EPR14639(2)] staining at 1/1000 dilution, shown in green;loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) staining at 1/20000 dilution, shown in red. In Western blot, ab197896 was shown to bind specifically to S100A4. A band was observed at 11 kDa in wild-type HeLa and A549 cell lysates with no signal observed at this size in S100A4 knockout HeLa cell line ab265709 (knockout cell lysate ab257046) and S100A4 knockout A549 cell line ab261865 (knockout cell lysate ab261674). To generate this image, wild-type and S100A4 knockout HeLa and S100A4 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

S100A4 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human S100A4 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-s100a4-knockout-hela-cell-line-ab265709'>ab265709</a>)

Lane 3:

Wild-type A549 cell lysate at 20 µg

Lane 4:

S100A4 knockout A549 cell lysate at 20 µg

Predicted band size: 12 kDa

Observed band size: 11 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling S100A4 with ab197896 at 1/200 (10 μg/mL). Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. ab150077, Alexa Fluor^® 488 Goat anti-Rabbit at 1/1000 (2 μg/mL) was used as the secondary antibody. Cells were counterstained with ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/mL). Nuclear counter stain was DAPI (blue).

Confocal image showing positive staining in HeLa cell line.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

Intracellular 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 Fluorr^® 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

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.

• IP

Lab

Immunoprecipitation - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

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.

All lanes:

Immunoprecipitation - Anti-S100A4 antibody [EPR14639(2)] (ab197896)

Predicted band size: 12 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

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

Compared with ab197896, ab124805 has higher sensitivity. We recommend ab124805 as an alternative for testing western blot.

We suggest optimizing experimental protocols (increasing lysate amount, using lower dilution or higher sensitivity ECL substrate) to improve results when using ab197896 in western blot.

All lanes:

Western blot - Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1:

Human tonsil tissue lysate at 20 µg

Lane 2:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 12 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

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:

Western blot - Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

S100A4 knockout HeLa cell lysate at 20 µg

Predicted band size: 12 kDa

Observed band size: 12 kDa

false

• WB

Lab

Western blot - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

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.

All lanes:

Western blot - Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1:

Wild-type A549 whole cell lysate at 20 µg

Lane 2:

S100A4 knockout A549 whole cell lysate at 20 µg

Lane 3:

A375 whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Predicted band size: 12 kDa

Observed band size: 12 kDa

false

• WB

Supplier Data

Western blot - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

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.

All lanes:

Western blot - Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1:

A549 (Human lung carcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 3:

Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Lane 4:

Mouse spleen tissue lysates at 20 µg

Lane 5:

Mouse bone marrow tissue lysates at 20 µg

Lane 6:

Mouse heart tissue lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 12 kDa

Observed band size: 12 kDa

false

Exposure time: 44s

• WB

Lab

Western blot - Anti-S100A4 antibody [EPR14639(2)] (AB197896)

Lanes 1 - 2 : Merged signal (red and green). Green - ab197896 observed at 11 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab197896 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 ab197896 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:

Western blot - Anti-S100A4 antibody [EPR14639(2)] (ab197896) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human S100A4 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-s100a4-knockout-hela-cell-lysate-ab257046'>ab257046</a>) at 20 µg

Predicted band size: 12 kDa

Observed band size: 11 kDa

false