Anti-Caspase-6/CASP-6 抗体 [EP1325Y]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caspase-6/CASP-6 antibody [EP1325Y] (AB52951)

ab52951 at 1 : 100 dilution staining Caspase-6/CASP-6 in human breast carcinoma by Immunohistochemistry Paraffin embedded tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Caspase-6/CASP-6 antibody [EP1325Y] (AB52951)

ab52951 at 1 : 100 dilution staining Caspase-6/CASP-6 in HeLa cells by Immunofluorescence.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Caspase-6/CASP-6 antibody [EP1325Y] (AB52951)

Overlay histogram showing Hela cells stained with ab52951 (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 (ab52951 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 >5000 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.

• WB

Lab

Western blot - Anti-Caspase-6/CASP-6 antibody [EP1325Y] (AB52951)

Western blot : Anti-Caspase-6/CASP-6 antibody [EP1325Y] ab52951 staining at 1/20000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 40 kDa in Wild-type HAP1 cell lysates with no signal observed at this size in CASP6 knockout HAP1 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Caspase-6/CASP-6 antibody [EP1325Y] (ab52951) at 1/20000 dilution

Lane 1:

Wild-type HAP1 cell lysates at 20 µg

Lane 2:

CASP6 knockout HAP1 cell lysates at 20 µg

Lane 3:

MCF7 cell lysates at 20 µg

Lane 4:

Jurkat cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 40 kDa

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• WB

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Western blot - Anti-Caspase-6/CASP-6 antibody [EP1325Y] (AB52951)

All lanes:

Western blot - Anti-Caspase-6/CASP-6 antibody [EP1325Y] (ab52951) at 1/20000 dilution

All lanes:

MCF-7 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated at 1/2000 dilution

Predicted band size: 33 kDa

Observed band size: 34 kDa

false