HRP Anti-Hsp27 antibody [EPR5477]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HRP Anti-Hsp27 antibody [EPR5477] (AB194079)

IHC image of Hsp27 staining in a section of formalin-fixed paraffin-embedded normal human skeletal muscle tissue*, performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab194079 at 1/500 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• WB

Lab

Western blot - HRP Anti-Hsp27 antibody [EPR5477] (AB194079)

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab194079 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

All lanes:

Western blot - HRP Anti-Hsp27 antibody [EPR5477] (ab194079) at 1/5000 dilution

All lanes:

Western blot - HeLa whole cell lysate (<a href='/en-us/products/cell-lysates/hela-whole-cell-lysate-ab150035'>ab150035</a>) at 10 µg

Predicted band size: 23 kDa

Observed band size: 27 kDa

true

Exposure time: 20s

• WB

Lab

Western blot - HRP Anti-Hsp27 antibody [EPR5477] (AB194079)

ab194079 was shown to specifically react with Hsp27 in wild-type HAP1 cells as signal was lost in HSPB1 (Hsp27) knockout cells. Wild-type and HSPB1 (Hsp27) knockout samples were subjected to SDS-PAGE. ab194079 and ab184095 (Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control (Alexa Fluor^® 680) loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/1000 dilution respectively. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.

All lanes:

Western blot - HRP Anti-Hsp27 antibody [EPR5477] (ab194079) at 1/5000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

HSPB1 (Hsp27) knockout HAP1 whole cell lysate at 20 µg

Predicted band size: 23 kDa

Observed band size: 27 kDa

false

Exposure time: 10s