All lanes : Anti-HSF1 antibody [EP1710Y] - ChIP Grade (ab52757) at 1/100000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Hsf1 knockout HAP1 whole cell lysate
Lane 3 : Hela whole cell lysate
Lane 4 : K562 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 57 kDa
Observed band size: 57 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab52757 observed at 57 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab52757 was shown to specifically react with in wild-type HAP1 cells as signal was lost in Hsf1 knockout cells. Wild-type and Hsf1 knockout samples were subjected to SDS-PAGE. Ab52757 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/100000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human ovarian carcinoma tissue sections labeling HSF1 with Purified ab52757 at 1:250 dilution (1.06 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

ab52757 (purified) at 1:20 dilution (2μg) immunoprecipitating HSF1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab52757 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab52727 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Chromatin was prepared from HeLa cells heat shocked (42^oC 30 minutes) according to the Abcam Dual X-ChIP protocol. Cells were fixed with EGS for 30 minutes, then formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab52757 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (sybr green approach).
Primers and probes are located in the first kb of the transcribed region.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling HSF1 with Purified ab52757 at 1:100 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1:200. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling HSF1 with purified ab52757 at 1:20 dilution (10 µg/ml) (red). Cells were fixed with 80% Methanol and permeabilised with 0.1% Tween-20. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Anti-HSF1 antibody [EP1710Y] - ChIP Grade (ab52757) at 1/50000 dilution (purified) + HeLa (Human cervix adenocarcinoma epithelial cell ) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 57 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST

Anti-HSF1 antibody [EP1710Y] - ChIP Grade (ab52757) at 1/100000 dilution (unpurified) + HeLa cell lysate at 10 µg

Secondary
HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 57 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling HSF1 with ab52757 at 1/1000 followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Nuclear staining on mouse testis. The section was incubated with ab52757 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), Ready to use.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling HSF1 with ab52757 at 1/1000 followed by a Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Nuclear staining on mouse colon. The section was incubated with ab52757 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101), Ready to use.

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

ab52757 staining HSF1 in wild-type Hap1 cells (top panel) and HSF1 knockout Hap1 cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab52757 at 1/250 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunocytochemistry/Immunofluorescence analysis of MCF-7 cells labelling HSF1 with unpurified ab52757 at 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody.
Control: PBS only.
Nuclear counter stain: DAPI.

Overlay histogram showing HeLa cells stained with unpurified ab52757 (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 (unpurified ab52757, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunohistochemical staining of paraffin-embedded human ovarian carcinoma using unpurified ab52757 at a 1:100 dilution.