Anti-Rab9 antibody [Mab9] - Late Endosome Marker

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab9 antibody [Mab9] - Late Endosome Marker (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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab9 antibody [Mab9] - Late Endosome Marker (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.

• IHC-P

AbReview35316****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Rab9 antibody [Mab9] - Late Endosome Marker (AB2810)

ab2810 staining Rab9 in Mouse adult testes tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with 4% paraformaldehyde and blocked with 5% BSA for 60 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100 in 1% BSA) for 15 hours at 4°C. A TRITC-conjugated Donkey anti-mouse IgG polyclonal (1/200) was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Qin Wen

• WB

Lab

Western blot - Anti-Rab9 antibody [Mab9] - Late Endosome Marker (AB2810)

Lanes 1-4 : Merged signal (red and green). Green - ab2810 observed at 25 kDa. Red - loading control ab181602 observed at 36 kDa.

ab2810 Anti-Rab9 antibody [Mab9] was shown to specifically react with Rab9 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265693 (knockout cell lysate ab257625) was used. Wild-type and Rab9 knockout samples were subjected to SDS-PAGE. ab2810 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Rab9 antibody [Mab9] - Late Endosome Marker (ab2810) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

RAB9A knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human RAB9A (Rab9) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-rab9a-rab9-knockout-hela-cell-line-ab265693'>ab265693</a>)

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

MDA-MB-231 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 23 kDa

Observed band size: 25 kDa

false

• WB

Project

Western blot - Anti-Rab9 antibody [Mab9] - Late Endosome Marker (AB2810)

All lanes:

Western blot - Anti-Rab9 antibody [Mab9] - Late Endosome Marker (ab2810) at 10 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Lane 3:

Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

Lane 4:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 5:

K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 23 kDa

Observed band size: 22 kDa,48 kDa,70 kDa

true

Exposure time: 20min