Immunohistochemical analysis of paraffin-embedded human colon tissue labeling ITLN1 + ITLN2 with ab252926 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in goblet cells of human colon (PMID: 22768319) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-ITLN1 + ITLN2 antibody [EPR22491-81] (ab252926) at 1/1000 dilution

Lane 1 : Rat colon tissue lysate
Lane 2 : Rat small intestine tissue lysate
Lane 3 : Mouse colon tissue 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: 35, 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

The actual band size higher than predicted mainly due to glycosylation. The molecular weight observed is consistent with what has been described in the literature (PMID: 11313366, PMID: 20628387). Lanes1-2 were developed using a higher sensitivity ECL substrate.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-2: 3 mins; Lane 3: 32 secs.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse colon tissue labeling ITLN1 + ITLN2 with ab252926 at 1/1250 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on goblet cells of mouse colon (PMID: 22768319) is observed. Counterstained with DAPI (blue).
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

All lanes : Anti-ITLN1 + ITLN2 antibody [EPR22491-81] (ab252926) at 1/1000 dilution

Lane 1 : Human small intestine tissue lysate
Lane 2 : PC-3 (human prostate adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : A549 (human lung carcinoma epithelial cell), whole cell lysate
Lane 4 : Caco-2 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 5 : Human fetal colon tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1 & 5 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 2-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 35, 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

The actual band size higher than predicted mainly due to glycosylation. The molecular weight observed is consistent with what has been described in the literature (PMID: 11313366, PMID: 20628387).

This blot was developed using a higher sensitivity ECL substrate.

Negative control: PC-3, A549, Caco-2 (PMID: 20628387).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1: 3 mins; Lane 2-5: 15 secs.

All lanes : Anti-ITLN1 + ITLN2 antibody [EPR22491-81] (ab252926) at 1/1000 dilution

Lane 1 : His-tagged human ITLN2 recombinant protein, 20 ng
Lane 2 : His-tagged human ITLN1 recombinant protein, 20 ng

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

Predicted band size: 35, 36 kDa


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat colon tissue labeling ITLN1 + ITLN2 with ab252926 at 1/1250 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on goblet cells of rat colon (PMID: 22768319) is observed. Counterstained with DAPI (blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling ITLN1 + ITLN2 with ab252926 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in goblet cells of rat colon (PMID: 22768319) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling ITLN1 + ITLN2 with ab252926 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in goblet cells of mouse colon (PMID: 22768319) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).