重组Anti-MARCKS抗体[EP1446Y]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MARCKS antibody [EP1446Y] (AB52616)

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 >5000 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] (AB52616)

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] (AB52616)

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

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MARCKS antibody [EP1446Y] (AB52616)

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] (AB52616)

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] (ab52616) at 1/20000 dilution

Lane 2:

Western blot - Anti-MARCKS antibody [EP1446Y] (ab52616) 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

• WB

CiteAb

Western blot - Anti-MARCKS antibody [EP1446Y] (AB52616)

MARCKS western blot using anti-MARCKS antibody [EP1446Y] ab52616. Publication image and figure legend from Rohrbach, T. D., Shah, N., et al., 2015, PLoS One, PubMed 26470026.

ab52616 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab52616 please see the product overview.

MARCKS localizes to the nucleus in D54 and U251 glioma cells.A) D54 and U251 cells were fixed and stained for MARCKS and DAPI with the merged image shown on the right side as indicated. B) D54 and U251 cell lysates were separated into nuclear and cytoplasmic fractions. The proteins were then were separated by 8% SDS-PAGE and probed for MARCKS, the cytoplasmic marker tubulin, and the nuclear marker, lamin.

false