Anti-PGAM5 antibody [RM1254]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PGAM5 antibody [RM1254] (AB322207)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling PGAM5 with ab322207 at 1/100 (4.85 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon carcinoma. The section was incubated with ab322207 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-PGAM5 antibody [RM1254] (AB322207)

Immunohistochemical analysis of paraffin-embedded A Wild-type HeLa (human cervix adenocarcinoma epithelial cell) cell pellet B PGAM5 knockout HeLa cell pellet tissue labeling PGAM5 with ab322207 at 1/100 (4.85 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) wild-type HeLa cell pellet, no staining on (B) PGAM5 knockout HeLa cell pellet. The section was incubated with ab322207 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-PGAM5 antibody [RM1254] (AB322207)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling PGAM5 with ab322207 at 1/100 (4.85 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. The section was incubated with ab322207 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

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Immunocytochemistry/ Immunofluorescence - Anti-PGAM5 antibody [RM1254] (AB322207)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PGAM5 KO HeLa (PGAM5 knockout human cervical adenocarcinoma epithelial cell) (ab265141) cells labelling PGAM5 with ab322207 at 1/100 (4.85 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/ 1000 2ug/ml dilution (Green).

Confocal image showing mitochondria staining in parental HeLa cells (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).

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 2ug/ml dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-PGAM5 antibody [RM1254] (AB322207)

PGAM5 was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab322207 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab322207 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 : ab322207 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab322207 in HeLa whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-PGAM5 antibody [RM1254] (ab322207) at 1/30 dilution

All lanes:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

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

false

Exposure time: 8s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PGAM5 antibody [RM1254] (AB322207)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling PGAM5 with ab322207 at 1/100 (4.85 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse testis. The section was incubated with ab322207 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-PGAM5 antibody [RM1254] (AB322207)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling PGAM5 with ab322207 at 1/100 (4.85 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat testis. The section was incubated with ab322207 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

Supplier Data

Western blot - Anti-PGAM5 antibody [RM1254] (AB322207)

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

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 22915595 and PMID : 30250224).

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

All lanes:

Western blot - Anti-PGAM5 antibody [RM1254] (ab322207) at 1/1000 dilution

Lane 1:

Untreated PANC-1(human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

PANC-1 treated with 100 uM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 2 hours, 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: 32 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-PGAM5 antibody [RM1254] (AB322207)

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

In Western blot, ab322207 was shown to bind specifically to PGAM5. Target of interest was observed at 32 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in PGAM5 knockout cell line (lane 2) (lane 2, knockout cell line ab265141 / knockout cell lysate ab257581).

Exposure time : Left panel : 180 seconds; right panel : 70 seconds

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

All lanes:

Western blot - Anti-PGAM5 antibody [RM1254] (ab322207) at 1/1000 dilution

Lane 1:

Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human PGAM5 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-pgam5-knockout-hela-cell-lysate-ab257581'>ab257581</a>) at 20 µg

Lane 3:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

MCF7 (human breast adenocarcinoma 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: 32 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-PGAM5 antibody [RM1254] (AB322207)

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

Lanes 1-2 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000. Lanes 3-6 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000.

Exposure time : Lanes 1-4 and 6 : 180 seconds; Lane 5 : 26 seconds

All lanes:

Western blot - Anti-PGAM5 antibody [RM1254] (ab322207) at 1/1000 dilution

Lane 1:

Human liver tissue lysate at 20 µg

Lane 2:

Human tonsil tissue lysate at 20 µg

Lane 3:

Mouse colon tissue lysate at 20 µg

Lane 4:

Mouse kidney tissue lysate at 20 µg

Lane 5:

Rat colon tissue lysate at 20 µg

Lane 6:

Rat liver tissue lysate at 20 µg

Secondary

Lanes 1 - 2:

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

Lanes 3 - 6:

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: 32 kDa

false

• WB

Supplier Data

Western blot - Anti-PGAM5 antibody [RM1254] (AB322207)

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

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

All lanes:

Western blot - Anti-PGAM5 antibody [RM1254] (ab322207) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 3:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma 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: 32 kDa,36 kDa

false

Exposure time: 180s