重组Anti-PYCR1抗体[EPR28646-7] (ab317563)

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

In Western blot, Anti-Vinculin antibody [EPR8185]  (ab129002) staining at 1/10000 dilution.

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

Low expression: lung, colon, breast, skeletal muscle.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control  (ab181602) staining at 1/200000 dilution.

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling PYCR1 with ab317563 at 1/100 (4.99 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human pancreas.
The section was incubated with ab317563 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) Human lung carcinoma. (B) Adjacent lung tissue. tissue labeling PYCR1 with ab317563 at 1/100 (4.99 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in (A) human lung carcinoma, almost no staining in (B) adjacent lung tissue.
The section was incubated with ab317563 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 breast carcinoma tissue labeling PYCR1 with ab317563 at 1/100 (4.99 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining in human breast carcinoma.
The section was incubated with ab317563 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 lung tissue labeling PYCR1 with ab317563 at 1/100 (4.99 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue: No staining in human lung.
The section was incubated with ab317563 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 HeLa (human cervical adenocarcinoma epithelial cell) cells labelling PYCR1 with ab317563 at 1/50 (9.98 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green).

Confocal image showing mitochondrial staining in HeLa cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

anti-COX IV mouse monoclonal antibody - Mitochondrial Marker was used to counterstain tubulin at 1/200 2.5 dilution (Magenta). 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 ug/ml dilution.

-ve control 1: ab317563 at 1/50 (9.98 ug/ml) dilution, followed by ab150120 at 1/1000 2 ug/mL dilution.
-ve control 2: anti-COX IV mouse monoclonal antibody 1/50 0.5ug/ml dilution, followed by ab150081 at 1/1000 2 ug/mL dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa(human cervical adenocarcinoma epithelial cell) cells labelling PYCR1 with ab317563 at 1/500 dilution (0.1 ug)/Red (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, ab150081) at 1/5000 dilution was used as the secondary antibody.

PYCR1 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab317563 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317563 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 2: ab317563 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab317563 in HeLa whole cell lysate

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

Exposure time: 3 seconds.

Dot blot analysis of PYCR1 using ab317563 at 1:1000 (0.499 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1:20,000 dilution.

Lane 1: His-tagged human PYCR1 recombinant protein
Lane 2: His-tagged human PYCR2 recombinant protein

Exposure time: 180 seconds.

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

This antibody does not cross-react with human PYCR2.

Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) as total protein control staining at 1/5000 dilution.