Anti-MARCKS antibody [EP1446Y] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MARCKS antibody [EP1446Y] - BSA and Azide free (AB247324)

This data was developed using ab52616, the same antibody clone in a different buffer formulation.Overlay histogram showing HeLa cells stained with ab52616 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab52616, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Hela cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-MARCKS antibody [EP1446Y] - BSA and Azide free (AB247324)

This data was developed using ab52616, the same antibody clone in a different buffer formulation.

ab52616 staining MARCKS in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 100% methanol and incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain.

Control : PBS only.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MARCKS antibody [EP1446Y] - BSA and Azide free (AB247324)

This data was developed using ab52616, the same antibody clone in a different buffer formulation.

ICC/IF image of ab52616 stained HeLa cells. The cells were 4% formaldehyde fixed (10 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 ab52616 at 1/200 dilution overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MARCKS antibody [EP1446Y] - BSA and Azide free (AB247324)

This data was developed using ab52616, the same antibody clone in a different buffer formulation.

ab52616 at 1/100 dilution staining human brain tissue; paraffin embedded.

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

• WB

Lab

Western blot - Anti-MARCKS antibody [EP1446Y] - BSA and Azide free (AB247324)

This data was developed using ab52616, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The molecular mass observed is consistent with what has been described in the literatures (PMID : 22619307, PMID : 8557118, PMID : 20224071).

An extra band might be detected at 100 kDa but we are unsure how to define it.

Lane 1:

Western blot - Anti-MARCKS antibody [EP1446Y] (<a href='/en-us/products/primary-antibodies/marcks-antibody-ep1446y-ab52616'>ab52616</a>) at 1/20000 dilution

Lane 2:

Western blot - Anti-MARCKS antibody [EP1446Y] (<a href='/en-us/products/primary-antibodies/marcks-antibody-ep1446y-ab52616'>ab52616</a>) at 1/1000 dilution

Lane 1:

Human brain lysate at 15 µg

Lane 2:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 15 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/20000 dilution

Predicted band size: 32 kDa

Observed band size: 80 kDa

false

Exposure time: 5s