Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

Immunohistochemical staining of paraffin embedded human thyroid carcinoma with purified ab176558 at a working dilution of 1/300. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human prostate tissue, labeling mtTFA using unpurified ab176558 at a 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

Immunofluorescence staining of MCF7 cells with purified ab176558 at a working dilution of 1/250, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab176558 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

Immunofluorescence analysis of MCF7 cells, labeling mtTFA using unpurified ab176558 at a 1/100 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human kidney tissue, labeling mtTFA using unpurified ab176558 at a 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• IP

Supplier Data

Immunoprecipitation - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

Western blot analysis on Immunoprecipitation pellet from either 1) 293T cell lysate, or 2) 1xPBS (negative control), showing mtTFA, using unpurified ab176558 at 1/10 dilution and HRP-conjugated anti-rabbit IgG preferentially detecting the non-reduced form of rabbit IgG.

All lanes:

Immunoprecipitation - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (ab176558)

Predicted band size: 29 kDa

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• IP

Unknown

Immunoprecipitation - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

ab176558 (purified) at 1/40 immunoprecipitating mtTFA in 10 μg HEK293 (Lanes 1 and 2, observed at 24 kDa). Lane 3 - PBS. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution. Blocking buffer and concentration : 5% NFDM/TBST Dilution buffer and concentration : 5% NFDM/TBST

Lane 1:

Immunoprecipitation - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (ab176558)

Lanes 1 - 2:

Immunoprecipitation - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (ab176558) at 1/40 dilution

Lane 1:

HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate at 10 µg

Lane 2:

ab176558 + HEK-293 whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab176558 in HEK-293 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 1/1000 dilution

Predicted band size: 29 kDa

false

• WB

Lab

Western blot - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (ab176558) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

MCF7 cell lysate at 10 µg

Lane 3:

HEK293 cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 29 kDa

Observed band size: 24 kDa

false

• WB

Supplier Data

Western blot - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (AB176558)

All lanes:

Western blot - Anti-mtTFA antibody [EPR12285] - Mitochondrial Marker (ab176558) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

A431 cell lysate at 10 µg

Lane 3:

MCF7 cell lysate at 10 µg

Lane 4:

293T cell lysate at 10 µg

Predicted band size: 29 kDa

true