All lanes : Anti-mtTFA antibody [EPR12285] (ab176558) at 1/10000 dilution (purified)

Lane 1 : HeLa cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : HEK293 cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 29 kDa
Observed band size: 24 kDa why is the actual band size different from the predicted?

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

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.

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.

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

All lanes : Anti-mtTFA antibody [EPR12285] (ab176558) at 1/10000 dilution (unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : A431 cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique.

Predicted band size: 29 kDa

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.

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.

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

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.