Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling SIGIRR with ab228977 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat kidney (PMID:14715412, 12925853). The section was incubated with ab228977 for 30 mins at room temperature. The immunostaining 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).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized MEF (mouse embryonic fibroblast (immortalized)) (Left) / Hepa1-6 (mouse hepatoma epithelial cell line) (Right) cells labelling SIGIRR with ab228977 at 1/600 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control: MEF (PMID 12925853).

IGFBP5 was immunoprecipitated from 0.35 mg Hepa1-6 (mouse hepatoma epithelial cell line) whole cell lysate 10μg with ab254324 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab228977 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: Hepa1-6 (mouse hepatoma epithelial cell) whole cell lysate 10μg

Lane 2: ab254324 IP in Hepa1-6 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab228977 in Hepa1-6 whole cell lysate.

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

Exposure time: 5 seconds.

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling SIGIRR with ab228977 at 1/1000 dilution  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse kidney (PMID:14715412, 12925853). The section was incubated with ab228977 for 30 mins at room temperature. The immunostaining 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).

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling SIGIRR with ab228977 at 1/1000 dilution  followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human colon (PMID:21077278) The section was incubated with ab228977 for 30 mins at room temperature. The immunostaining 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).

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling SIGIRR with ab228977 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human kidney (PMID:14715412, 12925853) The section was incubated with ab228977 for 30 mins at room temperature. The immunostaining 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-SIGIRR antibody [EPR22438-93] (ab228977) at 1/1000 dilution

Lane 1 : Mouse kidney tissue lysate
Lane 2 : Mouse kidney treated lysate treated with with PNGase F

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 46 kDa
Observed band size: 46, 80 kDa why is the actual band size different from the predicted?

The molecular weight observed is consistent with what has been described in the literature (PMID: 26344057).

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

Exposure time: 92 seconds.

All lanes : Anti-SIGIRR antibody [EPR22438-93] (ab228977) at 1/1000 dilution

Lane 1 : Human fetal kidney tissue lysate
Lane 2 : Rat liver tissue lysate
Lane 3 : Mouse kidney tissue lysate
Lane 4 : Mouse spleen tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution (VeriBlot for IP secondary antibody (HRP))
Lanes 2-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Predicted band size: 46 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

The different apparent molecular weights are due to different levels of glycosylation. (PMID: 26344057).

Exposure times.
Lane 1: 48 seconds; Lane 2: 7.75 seconds; Lane 3: 70 seconds; Lane 4: 3 minutes.

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

All lanes : Anti-SIGIRR antibody [EPR22438-93] (ab228977) at 1/1000 dilution

Lanes 1 & 5 : Hepa1-6 (mouse hepatoma epithelial cell), whole cell lysate
Lane 2 : MEF (immortalized mouse embryonic fibroblasts), whole cell lysate
Lane 3 : Mouse stomach tissue lysate
Lane 4 : Mouse pancreas tissue lysate
Lane 6 : Rat stomach tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1-4 & 6 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Lane 5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Human stomach tissue lysate)

Predicted band size: 46 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

The different apparent molecular weights are due to different levels of glycosylation. (PMID: 26344057).

Negative control: MEF (PMID 12925853)

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

Exposure times.

Lane 1: 15 seconds; Lanes 2-6: 3 minutes.