All lanes : Anti-Hsp60 antibody (ab46798) at 1/20000 dilution (purified)

Lane 1 : Mouse brain tissue lysate at 20 µg
Lane 2 : Mouse heart tissue lysate at 20 µg
Lane 3 : Mouse spleen tissue lysate at 20 µg
Lane 4 : Rat heart tissue lysate at 10 µg

Secondary
All lanes : HRP conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human lung adenocarcinoma with purified ab46798 at a working dilution of 1 in 100. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 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 analysis of HeLa cells labeling Hsp60 with ab46798 at a working dilution of 1 in 50 counter-stained with DAPI. The secondary antibody was ab150077 Alexa Fluor® 488 goat anti rabbit, used at a dilution of 1 in 500. Tubulin was stained with mouse anti-tubulin at a dilution of 1/1000 (ab7291) and Alexa Fluor® 594 goat anti-mouse at a dilution of 1/500 (ab150120) . The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in the bottom middle and right hand panels - for the first negative control, purified ab46798 was used at a dilution of 1/200 followed by an Alexa Fluor® 555 goat anti-mouse antibody at a dilution of 1/500 and for the second negative control mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab15007) were used.

ab46798 stained in Hela cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab46798 at 10 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

Overlay histogram showing HeLa cells fixed in 2% PFA and stained with purified ab46798 at a dilution of 1 in 150 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 150. Rabbit monoclonal IgG was used as an isotype control (black) and cells without incubation with the antibody were used as a negative control (blue line).

ab46798 (purified) at 1/40 immunoprecipitating Hsp60 in MCF7 (Lane 1). Lane 2 - PBS. For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

Anti-Hsp60 antibody (ab46798) at 1/20000 dilution (purified) + T47-D cell lysate at 10 µg

Secondary
HRP conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

All lanes : Anti-Hsp60 antibody (ab46798) at 1/50000 dilution (purified)

Lane 1 : MCF7 cell lysate
Lane 2 : SW480 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded rat kidney with purified ab46798 at a working dilution of 1 in 100. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 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.

Immunohistochemical staining of paraffin embedded mouse cardiac muscle with purified ab46798 at a working dilution of 1 in 100. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 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.

Immunohistochemical staining of paraffin embedded human cervical carcinoma with purified ab46798 at a working dilution of 1 in 100. The secondary antibody used is ab97051 Goat Anti-Rabbit IgG H&L (HRP) at a dilution of 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.

Unpurified ab46798 staining Hsp60 from human colon tissue by immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). Cells were formaldehyde fixed prior to blocking in 10% serum for 2 hours at 21°C. The primary antibody was diluted 1/500 and incubated with the sample for 2 hours at 21°C. Alexa fluor® 594 goat polyclonal, diluted 1/5000, was used as the secondary.

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Immunohistochemical analysis of paraffin-embedded human breast adenocarcinoma using unpurified ab46798 at 1/250 dilution.

Anti-Hsp60 antibody (ab46798) at 1/50000 dilution (purified) + Pig heart tissue lysate at 20 µg

Secondary
HRP conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 60 kDa
Observed band size: 60 kDa

Blocking Buffer: 5% NFDM/TBST

Dilution Buffer: 5% NFDM/TBST

Anti-Hsp60 antibody (ab46798) at 1/50000 dilution (unpurified) + T47D whole cell lysate (ab14899) at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/2000 dilution (Goat anti -rabbit HRP)

Predicted band size: 60 kDa
Observed band size: 60 kDa

Secondary antibody - anti-rabbit HRP (ab6721)