Flow Cytometry - Rab9 antibody [Mab9] (ab2810)

Overlay histogram showing THP1 cells stained with ab2810 (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 (ab2810, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. 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 THP1 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

Western blot - Rab9 antibody [Mab9] (ab2810)

All lanes : Anti-Rab9 antibody [Mab9] (ab2810) at 10 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 5 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 22 kDa
Observed band size : 22 kDa
Additional bands at : 48 kDa,70 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 20 minutes

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Rab9 antibody [Mab9] (ab2810)

ab2810 (2µg/ml) staining Rab9 in human spleen, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic and cell membrane staining.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 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.

Immunocytochemistry/ Immunofluorescence - Rab9 antibody [Mab9] (ab2810)

ab2810 staining Rab9 in MEF by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with PBST and blocked with 10% serum for 1 hour at room temperature. Samples were incubated with primary antibody (1/100 in PBS) for 8 hours at 4ºC. An Alexa Fluor^®488-conjugated Goat anti-mouse polyclonal (1/1000) was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Rab9 antibody [Mab9] (ab2810)

IHC image of Rab9 staining in human normal tonsil FFPE section, performed on a BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab2810, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX