Western blot - Rab5 antibody (ab18211)

Anti-Rab5 antibody - Early Endosome Marker (ab18211) at 1 µg/ml + Jurkat cell lysate at 20 µg

Secondary
Alexa Fluor Goat polyclonal to Rabbit IgG (700) at 1/5000 dilution

Predicted band size : 24 kDa
Observed band size : 24 kDa
Additional bands at : 40 kDa,70 kDa. We are unsure as to the identity of these extra bands.

Immunocytochemistry/ Immunofluorescence - Rab5 antibody - Early Endosome Marker (ab18211)

ICC/IF image of ab18211 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab18211, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

Western blot - Rab5 antibody - Early Endosome Marker (ab18211)

All lanes : Anti-Rab5 antibody - Early Endosome Marker (ab18211) at 1 µg/ml

Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab46770)
Lane 3 : Brain (Mouse) Tissue Lysate (ab27253)
Lane 4 : Testis (Mouse) Tissue Lysate - normal tissue
Lane 5 : Spinal Cord (Mouse) Tissue Lysate
Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 7 : Brain (Rat) Tissue Lysate - normal tissue
Lane 8 : Heart (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 24 kDa
Observed band size : 25 kDa (why is the actual band size different from the predicted?)

Immunohistochemistry (Frozen sections) - Rab5 antibody - Early Endosome Marker (ab18211)

ab18211 at 1/300 staining adult rat brain (perfusion fixed) tissue sections by IHC-Fr. Adult rat was perfused intracardially with paraformaldehyde 4% in PB 0.2M. The brain was post-fixed in the same fixative for 24 hours. Sections were cryoprotected with sucrose 20% and later frozen in OCT. Sections were incubated in free floating for 12h with the primary antibody (1/300) and later revealed with secondary antibody conjugated with Alexa Fluor ® 488 (1/2000).

The staining obtained is restricted to the cytoplasm and consists of a small and thin punctate staining. The picture shows the staining obtained at the level of the spinal cord using the X20 objective and zooming on two particular neurons.

This image is courtesy of an Abreview submitted by Dr Sophie Pezet

Immunoprecipitation - Rab5 antibody - Early Endosome Marker (ab18211)

ab18211 immunoprecipitated Rab5 in Mouse neuroblastoma N2a whole cell lysate. 10µg of cell lysate was incubaed with primary antibody (1/2000 in dilution buffer:0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 1% NP-40, 5% glycerol pH 7.4) for 2 hours at 22ºC and an AminoLink^® Plus Coupling Resin matrix.
For Western blotting an HRP conjugated goat monoclonal to rabbit IgG (1/2000) was used.

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Rab5 antibody - Early Endosome Marker (ab18211)

ICC/IF image of ab18211 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 (ab18211, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) 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.