All lanes : Anti-Hsp60 antibody [LK-1] (ab59457) at 0.05 µg/ml

Lane 1 : Rat Brain tissue lysates
Lane 2 : Rat Heart tissue lysates
Lane 3 : Rat Kidney tissue lysates
Lane 4 : Rat Liver tissue lysates
Lane 5 : Rat Lung tissue lysates
Lane 6 : Rat Pancreas tissue lysates
Lane 7 : Rat skeletal muscle tissue lysate
Lane 8 : Rat Spleen tissue lysate
Lane 9 : Rat Testes tissue lysate
Lane 10 : Rat Thymus tisuue lysate
Lane 11 : Cell lysates prepared from rat heart H9C2 cells
Lane 12 : Cell lysates prepared from mouse NIH3T3 cells
Lane 13 : Cell lysates prepared from mouse Pam212 cells

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat polyclonal to mouse IgG1 at 1/10 dilution

Predicted band size: 60 kDa

ICC/IF image of ab59457 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab59457, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Ab59457 staining human normal skin tissue. Staining is localised to mitochondria.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Ab59457 staining Hsp60 in Human malignant colon tissue sections by Immunohistochemistry. Antigen retrieval was by heat mediation using retrieval buffer, pH6. Cells were fixed with paraformaldehyde and blocked with 3% H₂O₂ for 10 minutes at 22°C. Samples were incubated with primary antibody at 1/2000 dilution for 2 hours at 22°C. A HRP conjugated goat polyclonal was used as a secondary antibody.

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Overlay histogram showing HeLa cells stained with ab59457 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab59457, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Hsp60 was immunoprecipitated using 0.5mg Rat Brain tissue lysate, 5µg of Mouse monoclonal to Hsp60 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Rat Brain tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab59457.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.
Band: 60kDa; Hsp60: non specific bands - 50kDa: We are unsure as to the identity of this extra band.