Anti-PTRF antibody [EPR27006-67]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PTRF antibody [EPR27006-67] (AB308623)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PTRF KO HeLa (PTRF knockout human cervical adenocarcinoma epithelial cel), ab265435 cells labelling PTRF with ab308623 at 1/500 (1.008 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 membranous and cytoplasmic staining in wildtype HeLa cells and showing no staining in PTRF knockout HeLa cells.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 2.5ug/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 ug/ml dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PTRF antibody [EPR27006-67] (AB308623)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling PTRF with ab308623 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on stromal cells of human kidney (PMID : 29065889). The section was incubated with ab308623 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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PTRF antibody [EPR27006-67] (AB308623)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labelling PTRF with ab308623 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on stromal cells of human liver (PMID : 33042738). The section was incubated with ab308623 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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PTRF antibody [EPR27006-67] (AB308623)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labelling PTRF with ab308623 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on stromal cells of human testis. The section was incubated with ab308623 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

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PTRF antibody [EPR27006-67] (AB308623)

Immunohistochemical analysis of paraffin-embedded Human ovarian carcin tissue labelling PTRF with ab308623 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on stromal cells of human ovarian carcinoma. The section was incubated with ab308623 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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PTRF antibody [EPR27006-67] (AB308623)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PTRF KO HeLa (ED030969, human PTRF knockout cervical adenocarcinoma epithelial cell, Left) / Parental HeLa (Right) cells labelling PTRF with ab308623 at 1/50 dilution (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PTRF antibody [EPR27006-67] (AB308623)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labelling PTRF with ab308623 at 1/2000 (0.252 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on stromal cells of rat testis (PMID : 27088550). The section was incubated with ab308623 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

• WB

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Western blot - Anti-PTRF antibody [EPR27006-67] (AB308623)

Blocking and diluting buffer and concentration : 5% NFDM/TBST. The blot of lane 1 was developed using a high sensitivity ECL substrate. In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : Lane 1 : 180 seconds, lane 2 : 59 seconds

All lanes:

Western blot - Anti-PTRF antibody [EPR27006-67] (ab308623) at 1/1000 dilution

Lane 1:

Human kidney tissue lysate at 20 µg

Lane 2:

U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

• WB

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Western blot - Anti-PTRF antibody [EPR27006-67] (AB308623)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of TBS. Lysates at 20 µg per lane. The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-PTRF antibody (ab308623) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab308623 was shown to bind specifically to PTRF. Target of interest was observed at 50 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in PTRF knockout cell line ab265435  (knockout cell lysate ab257620) (lane 2).  To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane.  Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C.  Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged.

Lane 1:

Wild-type HeLa whole cell lysate at 20 µg

Lane 2:

PTRF knockout HeLa whole cell lysate at 20 µg

Predicted band size: 43 kDa

Observed band size: 50 kDa

false