All lanes : Anti-MARCKS antibody [EP1446Y] (ab52616) at 1/1000 dilution (Purified)

Lane 1 : HeLa (human cervix adenocarcinoma) whole cell lysates prepared in RIPA lysis method
Lane 2 : HeLa (human cervix adenocarcinoma) whole cell lysates prepared in 1%SDS Hot lysis method

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 32 kDa
Observed band size: 87 kDa why is the actual band size different from the predicted?

We recommend to use 1%SDS Hot lysis prepare method to get desired WB results

Blocking/Diluting: Buffer and concentration 5% NFDM/TBST

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.

All lanes : Anti-MARCKS antibody [EP1446Y] (ab52616) at 1/1000 dilution (Purified)

Lane 1 : Human brain prepared in RIPA lysis method
Lane 2 : Human brain prepared in 1%SDS Hot lysis method

Secondary
All lanes : Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 32 kDa
Observed band size: 87 kDa why is the actual band size different from the predicted?

We recommend to use 1%SDS Hot lysis prepare method to get desired WB results

Blocking/Diluting Buffer and concentration: 5% NFDM/TBST

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 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. 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 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.

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.