Anti-ST14 抗体 [EPR26785-94] (ab307599)

All lanes : Anti-ST14 antibody [EPR26785-94] (ab307599) at 1/1000 dilution

Lane 1 : NAMALWA (human Burkitt's lymphoma b lymphocyte) whole cell lysate 40 µg
Lane 2 : T-47D (human ductal breast epithelial tumor epithelial cell), whole cell lysate 40 µg
Lane 3 : MDA-MB-231 (human breast adenocarcinoma epithelial cell) whole cell lysate 40 µg
Lane 4 : HaCaT (human skin keratinocyte) whole cell lysate 40 µg
Lane 5 : Mouse kidney tissue lysate 40 µg

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

Predicted band size: 94 kDa
Observed band size: 80-95 kDa why is the actual band size different from the predicted?

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

Negative control: MDA-MB-231(PMID:28915606). The lane 5 was developed using a high sensitivity ECL substrate. The identity of the lower MW bands beneath 80 kDa are expected to be degraded ST14.

Exposure time:

Lanes 1, 3 and 4: 180 seconds

Lane 2 : 70 seconds

Lane 5 : 180 seconds

All lanes : Anti-ST14 antibody [EPR26785-94] (ab307599) at 1/1000 dilution

Lane 1 : Rat kidney tissue lysate 40 µg
Lane 2 : NR8383 (rat alveolar macrophage) whole cell lysate 20 µg

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

Predicted band size: 94 kDa
Observed band size: 80-95 kDa why is the actual band size different from the predicted?

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

The blot was developed using a high sensitivity ECL substrate. The identity of the lower MW bands beneath 80 kDa are expected to be degraded ST14.

Exposure time:

Lanes 1: 59 seconds

Lane 2 : 70 seconds

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling ST14 with ab307599 at 1/100 (4.94 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human colon (PMID: 22081509). The section was incubated with ab307599 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling ST14 with ab307599 at 1/100 (4.94 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse colon. The section was incubated with ab307599 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling ST14 with ab307599 at 1/100 (4.94 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on rat colon. The section was incubated with ab307599 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded (A) T-47D (human ductal breast epithelial tumor epithelial cell) cell pellet tissue labeling ST14 with ab307599 at 1/100 (4.94 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on (A) T-47D cell pellet, no staining on (B) MDA-MB-231 cell pellet. The section was incubated with ab307599 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling ST14 with ab307599 at 1/100 (4.94 µg/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Negative control: no staining on human testis (PMID:12871983). The section was incubated with ab307599 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized T-47D (human ductal breast epithelial tumor epithelial cell) cells labelling ST14 with ab307599 at 1/50 (9.88 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing membranous staining in T-47D cell line, no staining was observed in MDA-MB-231 cell line.

Negative control: MDA-MB-231(PMID:28915606). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.