All lanes : Anti-SOCS3 antibody [EPR24090-74] (ab280884) at 1/1000 dilution

Lane 1 : Untreated RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 2 : RAW264.7 treated with 100ng/ml LPS(Lipopolysaccharide) for 4 hours, whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 24 kDa
Observed band size: 26 kDa why is the actual band size different from the predicted?

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:17114447, 32306216, 31238931)

Exposure time: 26 seconds

 

All lanes : Anti-SOCS3 antibody [EPR24090-74] (ab280884) at 1/1000 dilution

Lane 1 : Human colon tissue lysate
Lane 2 : Human placenta tissue lysate
Lane 3 : Human skin tissue lysate
Lane 4 : Mouse spleen tissue lysate
Lane 5 : Mouse placenta tissue lysate
Lane 6 : Mouse skin tissue lysate
Lane 7 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 24 kDa
Observed band size: 23,26 kDa why is the actual band size different from the predicted?

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID:17114447, 32306216, 31238931)
SOCS3 exists in two isoforms of different length that are encoded by one gene
Negative control: skin (PMID: 22739986)

 Exposure time: 3 minutes

 

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling SOCS3 with ab280884 at 1/1000 (0.576 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the stromal cells in human colon. (PMID: 28603075) The section was incubated with ab280884 for 30 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labelling SOCS3 with ab280884 at 1/1000 (0.576 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human placenta. (PMID: 29201185 ) The section was incubated with ab280884 for 30 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labelling SOCS3 with ab280884 at 1/1000 (0.576 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse spleen. The section was incubated with ab280884 for 30 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labelling SOCS3 with ab280884 at 1/1000 (0.576 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on the stromal cells in rat colon. The section was incubated with ab280884 for 30 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 Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

Immunohistochemical analysis of paraffin-embedded Human skin tissue labelling SOCS3 with ab280884 at 1/1000 (0.576 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: no staining on human skin. (PMID: 22739986) The section was incubated with ab280884 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counter stain Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

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

SOCS3 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug with ab280884 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280884 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab280884 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG1 (ab18443) instead of ab280884 in HeLa whole cell lysate

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

Exposure time: 58 seconds.

 

SOCS3 was immunoprecipitated from 0.35 mg RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 100 ng/ml LPS (Lipopolysaccharide) for 4 hours whole cell lysate 10 ug with ab280884 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280884 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) treated with 100 ng/ml LPS (Lipopolysaccharide) for 4 hours whole cell lysate 10 ug

Lane 2: ab280884 IP in RAW 264.7 treated with 100 ng/ml LPS (Lipopolysaccharide) for 4 hours whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab280884 in treated with 100 ng/ml LPS (Lipopolysaccharide) for 4 hours whole cell lysate

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

Exposure time: 3 minutes.