Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Extracellular matrix protein 1 with ab253185 at 1/5000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). The section was incubated with ab253185 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

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

 Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse live tissue labeling Extracellular matrix protein 1 with ab253185 at 1/5000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). The section was incubated with ab253185 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

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

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Extracellular matrix protein 1 with ab253185 at 1/5000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer). The section was incubated with ab253185 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

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

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

All lanes : Anti-Extracellular matrix protein 1 antibody [EPR22411-279] (ab253185) at 1/1000 dilution

Lane 1 : Wild-type mouse liver lysate
Lane 2 : ECM1 knockout mouse liver lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 63 kDa
Observed band size: 48,85 kDa why is the actual band size different from the predicted?

The wild-type and ECM1 knockout mouse liver lysates were kindly provided by an anonymous collaborator.

ab253185 was shown to specifically react with Extracellular matrix protein 1 in wild-type mouse liver as signal was lost in ECM1 knockout liver. Wild-type and ECM1 knockout samples were subjected to SDS-PAGE. ab253185 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 1/50,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

Exposure time 7.75 secs.

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Extracellular matrix protein 1 antibody [EPR22411-279] (ab253185) at 1/1000 dilution

Lane 1 : 4T1 (mouse mammary gland carcinoma epithelial cell), whole cell lysate
Lane 2 : LLC1 (mouse lung carcinoma), whole cell lysate
Lane 3 : Hepa1-6 (mouse hepatoma epithelial cell), whole cell lysate
Lane 4 : Mouse skin lysate
Lane 5 : Mouse liver lysate
Lane 6 : Rat skin lysate
Lane 7 : Rat liver lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 63 kDa
Observed band size: 48,85 kDa why is the actual band size different from the predicted?

Mouse ECM1 has two isoforms: A long isoform reported to be 85 kDa, and the shorter form 48 kDa.

The molecular weight observed is consistent with what has been described in the literature (PMID: 7608209; PMID: 23202415).

Exposure times: Lanes 1-2: 7.75 seconds; Lane 3: 1 second; Lanes 4-7: 10 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

Extracellular matrix protein 1 was immunoprecipitated from 0.35 mg mouse lung lysate with ab253185 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab253185 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse lung lysate 10µg.

Lane 2: ab253185 IP in mouse lung lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab253185 in mouse lung lysate.

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

Exposure time: 30 seconds.

 

Extracellular matrix protein 1 was immunoprecipitated from 0.35 mg mouse liver lysate with ab253185 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab253185 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: Mouse liver lysate 10µg.

Lane 2: ab253185 IP in mouse liver lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab253185 in mouse liver lysate.

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

Exposure time: 3 mins.

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized 4T1 (mouse mammary gland carcinoma epithelial cell) cells labeling Extracellular matrix protein 1 with ab253185 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.