Anti-Fragilis antibody

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fragilis antibody (AB15592)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling Fragilis with ab320853 at 1/2000 (0.245 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human kidney in (A) Anti-Fragilis antibody ab320853 (recombinant multiclonal ) and (B) Anti-Fragilis antibody ab15592 (polyclonal antibody). The section was incubated with ab320853 or ab15592 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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Fragilis antibody (AB15592)

ab15592 staining Fragilis in mES cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab15592 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• IHC-Fr

Collaborator

Immunohistochemistry (Frozen sections) - Anti-Fragilis antibody (AB15592)

IHC showing localisation of Fragilis (red) in cryosections of male genital ridges of E12.5 mouse embryos. Germ cells can be identified by staining with anti-Oct4 (green).

ab15592 (anti-Fragilis) = red
Oct4 = green
DAPI (DNA) = blue

The dilution of ab15592 used was 10 ug/ml.

This image is courtesy of Petra Hajkova, University of Cambridge

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fragilis antibody (AB15592)

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling Fragilis with ab320853 at 1/4000 (0.123 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat lung in (A) Anti-Fragilis antibody ab320853 (recombinant multiclonal ) and (B) Anti-Fragilis antibody ab15592 (polyclonal antibody). The section was incubated with ab320853 or ab15592 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Fragilis antibody (AB15592)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling Fragilis with ab320853 at 1/4000 (0.123 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse lung in (A) Anti-Fragilis antibody ab320853 (recombinant multiclonal )and (B) Anti-Fragilis antibody ab15592 (polyclonal antibody). The section was incubated with ab320853 or ab15592 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

• IP

Unknown

Immunoprecipitation - Anti-Fragilis antibody (AB15592)

Fragilis - Primordial Germ Cell Marker was immunoprecipitated using 0.5mg E14Tg2a whole cell extract, 5µg of Rabbit polyclonal to Fragilis and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, E14Tg2a whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab15592.
Secondary : Clean blot (HRP conjugate) at 1/1000 dilution.
Band : 13kDa : Fragilis - Primordial Germ Cell Marker.

All lanes:

Immunoprecipitation - Anti-Fragilis antibody (ab15592)

Predicted band size: 15 kDa

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• WB

Supplier Data

Western blot - Anti-Fragilis antibody (AB15592)

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

Negative control : Jurkat and 293T (PMID : 21177806)

Anti-Fragilis antibody (ab320853, recombinant monoclonal) showed less background than Anti-Fragilis antibody (ab15592, polyclonal) with human samples.

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

Exposure time : Recombinant multiclonal ab320853 : 70 seconds Polyclonal ab15592 : 103 seconds

All lanes:

Western blot - Anti-Fragilis antibody [RM1224] (<a href='/en-us/products/primary-antibodies/fragilis-antibody-rm1224-ab320853'>ab320853</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 3:

Jurkat (human T cell leukemia T lymphocyte from peripheral blood) whole cell lysate at 20 µg

Lane 4:

293T (human embryonic kidney 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

Observed band size: 36 kDa,15 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Fragilis antibody (AB15592)

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

Low expression : A20

Anti-Fragilis antibody (ab320853, recombinant monoclonal) showed less background than Anti-Fragilis antibody (ab15592, polyclonal) with mouse samples.

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

Recombinant multiclonal ab320853 : 26 seconds Polyclonal ab15592 : 70 seconds

All lanes:

Western blot - Anti-Fragilis antibody [RM1224] (<a href='/en-us/products/primary-antibodies/fragilis-antibody-rm1224-ab320853'>ab320853</a>) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 2:

A20 (mouse reticum sarcoma B lymphocyte) 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

Observed band size: 36 kDa,15 kDa

false

Exposure time: 15s

• WB

Project

Western blot - Anti-Fragilis antibody (AB15592)

All lanes:

Western blot - Anti-Fragilis antibody (ab15592) at 1 µg/mL

Lane 1:

Mouse Embryonic Stem Cell lysate at 10 µg

Lane 2:

Mouse Embryonic Germ Cell lysate at 10 µg

Lane 3:

Mouse Embryonic Stem Cell lysate at 10 µg with Fragilis peptide (<a href='/en-us/products/unavailable/fragilis-peptide-ab15737'>ab15737</a>)

Lane 4:

Mouse Embryonic Germ Cell lysate at 10 µg with Fragilis peptide (<a href='/en-us/products/unavailable/fragilis-peptide-ab15737'>ab15737</a>)

Predicted band size: 15 kDa

Observed band size: 13 kDa

false

• WB

CiteAb

Western blot - Anti-Fragilis antibody (AB15592)

Fragilis western blot using anti-Fragilis antibody ab15592. Publication image and figure legend from Lu, Z., Wu, M., et al., 2015, Stem Cells Int, PubMed 26635882.

ab15592 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab15592 please see the product overview.

Morphology and characteristics of the established OSCs. (a) Overview of the OSCs immediately after MACS (left) and the established OSCs (right) which formed the typical structure of cell clusters. (b) Reverse transcriptional PCR analysis for expression profile of OSCs and ovarian tissues as the positive control. There were two set of genes : one for oocytes including Gdf9, Nobox, and Zp3 and another one for germ cells including Fragilis, MVH, Prdm1, and Tert, displaying that OSCs are characteristic of germ cells. No RT, PCR of RNA sample without reverse transcription. (c) Immunofluorescence for MVH and Fragilis in established OSCs and STO as negative control. Green, MVH, and Fragilis immunofluorescence; Blue, DAPI. At the bottom are the images (bright field, Green, DAPI, and merge) of STO from left to right. (d) Alkaline phosphatase staining for established OSCs, mESCs, and STO showed that OSCs (arrows) were positive, whereas mESCs were strongly positive. mESCs, mouse embryonic stem cells. (e-f) Cytogenetic analysis for established OSCs showed normal karyotype.

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