Anti-Mitofilin antibody [EPR8749]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mitofilin antibody [EPR8749] (AB137057)

Immunohistochemical analysis of paraffin embedded Human testis tissue labelling Mitofilin with ab137057 (unpurified) at 1/100 dilution.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mitofilin antibody [EPR8749] (AB137057)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human hepatocellular cancer tissue sections labeling Mitofilin with purified ab137057 at 1/250 dilution (0.508 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mitofilin antibody [EPR8749] (AB137057)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human thyroid cancer tissue sections labeling Mitofilin with purified ab137057 at 1/250 dilution (0.508 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Mitofilin antibody [EPR8749] (AB137057)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Mitofilin with purified ab137057 at 1/100 dilution (1.3 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Mitofilin antibody [EPR8749] (AB137057)

Immunohistochemical analysis of paraffin embedded Human colon tissue labelling Mitofilin with ab137057 (unpurified) at 1/100 dilution.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Mitofilin antibody [EPR8749] (AB137057)

Immunofluorescent staining of HeLa cells labelling Mitofilin with ab137057 (unpurified) at 1/250 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Mitofilin antibody [EPR8749] (AB137057)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Mitofilin with purified ab137057 at 1/20 dilution (10μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• WB

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Western blot - Anti-Mitofilin antibody [EPR8749] (AB137057)

All lanes:

Western blot - Anti-Mitofilin antibody [EPR8749] (ab137057) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

Human fetal heart tissue lysate at 10 µg

Lane 3:

Human fetal muscle tissue lysate at 10 µg

Lane 4:

A673 cell lysate at 10 µg

Lane 5:

Saos 2 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

Predicted band size: 84 kDa

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• WB

Lab

Western blot - Anti-Mitofilin antibody [EPR8749] (AB137057)

Lanes 1 - 4 : Merged signal (red and green). Green - ab137057 observed at 84 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab137057 was shown to specifically react with IMMT in wild-type HAP1 cells as signal was lost in IMMT knockout cells. Wild-type and IMMT knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab137057 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Mitofilin antibody [EPR8749] (ab137057) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

IMMT knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HepG2 whole cell lysate at 20 µg

Lane 4:

Human Heart whole cell lysate at 20 µg

Predicted band size: 84 kDa

Observed band size: 84 kDa

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• WB

Lab

Western blot - Anti-Mitofilin antibody [EPR8749] (AB137057)

All lanes:

Western blot - Anti-Mitofilin antibody [EPR8749] (ab137057) at 1/10000 dilution

Lane 1:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Human fetal heart lysates at 20 µg

Lane 3:

Human skeletal muscle lysates at 20 µg

Secondary

All lanes:

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

Predicted band size: 84 kDa

Observed band size: 84 kDa

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• WB

CiteAb

Western blot - Anti-Mitofilin antibody [EPR8749] (AB137057)

Mitofilin western blot using anti-Mitofilin antibody [EPR8749] ab137057. Publication image and figure legend from Shaiken, T. E. & Opekun, A. R., 2014, Sci Rep, PubMed 24815916.

ab137057 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 ab137057 please see the product overview.

Patterns of protein distribution in HeLa cells.Cyt. Cont is a control for cytosolic proteins obtained with the 0.3% Chaps buffer cell lysis (far left panel of bands) for the CDS method; Nuc. Cont is a control for nuclear proteins obtained with the classical method of nuclei isolation in hypotonic buffer (far right panel of bands) for the CDS method. (A) Proteins of cytosol : proteins extracted by regular lysis buffer and Buffer A from the cytoplasm. They are not detected in the perinuclear and the nuclear fractions. Nuclear fractions were obtained with new and classical nuclei extraction techniques (B) Proteins detected in the cytosol and the perinuclear fraction : proteins were detected as cytosolic proteins with both cellular lysis technique; in addition, these proteins also appeared in the perinuclear fraction by extraction with Buffer B. (C) Proteins of perinuclear fraction : proteins are detected only in perinuclear fraction by buffer B extraction. p53 protein was detected with long exposure. (D) Proteins detected in the nuclear and the perinuclear fractions : transcription factor CREB appeared in both fractions. (E) Nuclear proteins : proteins were detected in nuclear fraction. Nuclear proteins were obtained with new and classical nuclei isolation techniques. The PVDF membranes were cropped into two halves and the high and low molecular weight proteins were shown correspondingly.

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