Anti-Progranulin antibody [MJF-D22103-42R]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling with ab327420 at 1/2000 (0.248 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on human breast carcinoma. The primary antibody was incubated 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-Progranulin antibody [MJF-D22103-42R] (AB327420)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A431 (human epidermoid carcinoma epithelial cell) labelling with ab327420 at 1/50 (9.92 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in A431 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Parental HEK293T (human embryonic kidney epithelial cell) and GRN KO HEK293T (ab266739) cells labelling with ab327420 at 1/50 (9.92 μg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic staining in parental HEK293T cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling with ab327420 at 1/2000 (0.248 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on human placenta. The primary antibody was incubated 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Immunohistochemical analysis of paraffin-embedded Human gastric carcinoma tissue labeling with ab327420 at 1/2000 (0.248 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on human gastric carcinoma. The primary antibody was incubated 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling with ab327420 at 1/2000 (0.248 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive on human spleen. The primary antibody was incubated 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle tissue labeling with ab327420 at 1/2000 (0.248 μg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : almost no staining on human skeletal muscle. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

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

• IP

Lab

Immunoprecipitation - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Progranulin was immunoprecipitated from 0.35 mg HEK293T (human embryonic kidney epithelial cell) whole cell lysate with ab327420 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab327420 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

The band beneath the target band (74 kDa) in lane 2 is likely to be isoform of Progranulin.

All lanes:

Immunoprecipitation - Anti-Progranulin antibody [MJF-D22103-42R] (ab327420) at 1/1000 dilution

Lane 1:

Wild-type HEK293T (human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

Wild-type HEK293T (human embryonic kidney epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab327420 in parental HEK293T whole cell lysate

Lane 4:

Progranulin knockout HEK293T whole cell lysate at 10 µg

Lane 5:

Progranulin knockout HEK293T whole cell lysate

Lane 6:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab327420 in DNMT3A knockout HeLa whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Observed band size: 74 kDa

false

Exposure time: 84s

• IP

Lab

Immunoprecipitation - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Progranulin was immunoprecipitated from 0.35 mg A431 (human epidermoid carcinoma epithelial cell) whole cell fresh lysate with ab327420 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab327420 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : A431 (human epidermoid carcinoma epithelial cell) whole cell fresh lysate
Lane 2 : ab327420 IP in A431 (human epidermoid carcinoma epithelial cell) whole cell fresh lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab327420 in A431 whole cell fresh lysate

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

Exposure time : 58 seconds.

The band beneath the target band (74 kDa) is likely to be isoform of Progranulin.

All lanes:

Immunoprecipitation - Anti-Progranulin antibody [MJF-D22103-42R] (ab327420) at 1/1000 dilution

Lane 1:

A431 (human epidermoid carcinoma epithelial cell) whole cell fresh lysate at 10 µg

Lane 2:

ab327420 at 1/30 IP in A431 (human epidermoid carcinoma epithelial cell) whole cell fresh lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab327420 in A431 whole cell fresh lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 74 kDa

false

Exposure time: 58s

• IP

Lab

Immunoprecipitation - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

Progranulin was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) whole cell fresh lysate with ab327420 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab327420 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T (human embryonic kidney epithelial cell) whole cell fresh lysate
Lane 2 : ab327420 IP in 293T (human embryonic kidney epithelial cell) whole cell fresh lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab327420 in 293T whole cell fresh lysate

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

Exposure time : 180 seconds.

The band beneath the target band (74 kDa) is likely to be isoform of Progranulin. This blot was developed using a high-sensitivity ECL substrate, allowing for the detection of proteins in the mid-femtogram range.

All lanes:

Immunoprecipitation - Anti-Progranulin antibody [MJF-D22103-42R] (ab327420) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell fresh lysate at 10 µg

Lane 2:

ab327420 at 1/30 IP in 293T (human embryonic kidney epithelial cell) whole cell fresh lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab327420 in 293T whole cell fresh lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 74 kDa

true

Exposure time: 180s

• WB

Lab

Western blot - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

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

The band beneath the target band (74 kDa) in lane 4 is likely to be isoform of Progranulin.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1 : 59 seconds; Lanes 2-3 : 180 seconds; Lane 4 : 92 seconds

All lanes:

Western blot - Anti-Progranulin antibody [MJF-D22103-42R] (ab327420) at 1/1000 dilution

Lane 1:

Human breast cancer tissue lysate at 20 µg

Lane 2:

Human liver tissue lysate at 20 µg

Lane 3:

Human kidney tissue lysate at 20 µg

Lane 4:

Human spleen tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 74 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-Progranulin antibody [MJF-D22103-42R] (AB327420)

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

In Western blot, ab327420 was shown to bind specifically to Progranulin. Target of interest was observed at 74 KDa in wild-type HEK293T cell lysates (lane 1) with no signal observed at these sizes in Progranulin knockout cell line (lane 2)(lane 2, knockout cell line ab266738 / knockout cell lysate ab257235).

The band beneath the target band (74 kDa) in lane 4 is likely to be isoform of Progranulin.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 180 seconds; Lanes 3-4 : 26 seconds

All lanes:

Western blot - Anti-Progranulin antibody [MJF-D22103-42R] (ab327420) at 1/1000 dilution

Lane 1:

Wild-type HEK293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Progranulin knockout HEK293T whole cell lysate at 20 µg

Lane 3:

A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 74 kDa,36 kDa

false