Anti-HuD 抗体 [EPR26472-54] (BSA and Azide free) (ab302515)

All lanes : Anti-HuD antibody [EPR26472-54] (ab302514) at 1/1000 dilution

Lane 1 : SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate
Lane 2 : K-562 (human chronic myelogenous leukemia lymphoblast), 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: 42 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

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

Negative control: K-562 (PMID: 25523825).

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling HuD with ab302514 at 1/500 dilution (1.054 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Positive staining on human cerebrum (PMID:20584986). The section was incubated with ab302514 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labeling HuD with ab302514 at 1/500 dilution (1.054 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Positive staining on human cerebellum. The section was incubated with ab302514 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human pancreas tissue labeling HuD with ab302514 at 1/500 dilution (1.054 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Positive staining on islet in human pancreas at a low level (PMID:22387028). The section was incubated with ab302514 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human glioma tissue labeling HuD with ab302514 at 1/500 dilution (1.054 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Positive staining on human glioma. The section was incubated with ab302514 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 Leica DS9800 (BOND™ Polymer Refine Detection kit).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded HEK-293T cell pellets. ab302514 labeling HuD at 1/500 dilution (0.105 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection kit). Positive staining on HEK-293T cells transfected with a human HuD expression vector (Panel A); No staining on HEK-293T cells transfected with a human HuC expression vector (Panel B); No staining on HEK-293T cells transfected with a human HuB expression vector (Panel C); No staining on HEK-293T cells transfected with an empty vector (Panel D). The section was incubated with ab302514 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 was used.

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SH-SY5Y (human neuroblastoma epithelial cell) cells labeling HuD with AB302514 at 1/500 dilution (1.054 µ/mL), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preabsorbed antibody at 1/1000 dilution (2µ/mL) (Green). Confocal image showing cytoplasmic and nuclear staining in SH-SY5Y cell line. Negative control: K-562 (PMID: 25523825) . ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (2.5µ/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody control: Primary diluent was used instead of primary antibody, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preabsorbed at 1/1000 dilution (2µ/mL).

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

Flow cytometric (Intracellular) analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized K562 (human chronic myelogenous leukemia lymphoblast, Left) / SH-SY5Y (human neuroblastoma epithelial cell, Right) cells labeling HuD with AB302514 at 1/500 dilution (0.1 µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control: K-562 (PMID: 25523825).

This data was developed using ab302514, the same antibody clone in a different buffer formulation.

HuD was immunoprecipitated from 0.35 mg SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate 10 µg with AB302514 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using AB302514 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate 10 µg (Inset)

Lane 2: AB302514 IP in SH-SY5Y whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of AB302514 in SH-SY5Y whole cell lysate

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

Exposure time: 15 seconds