Anti-PON1 antibody [17A12] (ab24261)

  Western blot - PON1 antibody [17A12] (ab24261)

All lanes : Anti-PON1 antibody [17A12] (ab24261) at 1/500 dilution

Lane 1 : HeLa cell lysates.
Lane 2 : HepG2 cell lysates.


Predicted band size : 40 kDa

  Western blot

Anti-PON1 antibody [17A12] (ab24261) at 1/500 dilution + PON1 protein (ab53376) at 0.01 µg

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/500 dilution
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 1 minute

  Immunocytochemistry/ Immunofluorescence-PON1 antibody [17A12](ab24261)

ICC/IF image of ab24261 stained HeLa cells. The cells were 4% PFA 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 (ab24261, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)-PON1 antibody [17A12](ab24261)

ab24261 (1µg/ml) staining PON1 in human liver using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining of hepatocytes.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

  Flow Cytometry - PON1 antibody [17A12] (ab24261)

Overlay histogram showing HepG2 cells stained with ab24261 (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 (ab24261, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 100% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.