Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on the base of the crypts of human colon (PMID: 24151532) 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).

Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/1000 dilution

Lane 1 : HT-1080 (human fibrosarcoma epithelial cell), whole cell lysate
Lane 2 : Human brain tissue lysate
Lane 3 : Mouse brain tissue lysate
Lane 4 : Rat stomach tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lanes 1 & 3-4 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 2 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 117 kDa
Observed band size: 130 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: 21508957).

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1: 70 seconds; Lanes 2- 4: 3 minutes.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse colon tissue labeling Eph receptor B2 with ab252935 at 1/250 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on the membrane of epithelial cells, especially located in the basal crypt region (PMID: 19621336) 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-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/5000 dilution

Lane 1 : His-tagged human EPHB1 recombinant protein, 10ng
Lane 2 : His-tagged human EPHB2 recombinant protein, 10 ng
Lane 3 : His-tagged human EPHB3 recombinant protein, 10 ng

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: 117 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?

Exposure time: Lane 1: 3 minutes; Lane 2: 3.25 seconds; Lane 3: 3 minutes

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

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on rat colon (PMID: 24151532) 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).

Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat colon tissue labeling Eph receptor B2 with ab252935 at 1/250 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Positive staining on the membrane of epithelial cells, especially located in the basal crypt region (PMID: 19621336) 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).

Lane 1 : Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/5000 dilution
Lane 2 : Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/1000 dilution

Lane 1 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 2 : 4T1 (mouse mammary gland carcinoma epithelial cell), whole cell 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: 117 kDa
Observed band size: 130 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: 21508957).

Exposure times: Lane 1: 3 minutes; Lane 2: 37 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on mouse colon (PMID: 24151532) 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).

Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human bladder cancer tissue labeling Eph receptor B2 with ab252935 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on human bladder cancer (PMID: 24151532, 25148033) 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).

Heated citrate solution (10mM citrate pH 6.0 + 0.05% Tween-20).

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse liver tissue labeling Eph receptor B2 with ab252935 at 1/50 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. No staining in mouse liver. Negative control: liver (PMID: 19366806). 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).

Anti-Eph receptor B2 antibody [EPR22427-268] (ab252935) at 1/1000 dilution + Rat brain tissue lysate at 20 µg

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

Predicted band size: 117 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

The upper band around 130 kDa is full length EPHB2 which is consistent to previous report (PMID: 21508957), while the lower band around 80 kDa could be non-specific band or cleaved fragment (PMID: 18713744). The expression on rat brain tissue is consistent with what has been described in the literature (PMID: 16840724).

This blot was developed using a higher sensitivity ECL substrate.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat liver tissue labeling Eph receptor B2 with ab252935 at 1/50 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. No staining in rat liver. Negative control: liver (PMID: 19366806). 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).