Anti-Rab5 antibody - Early Endosome Marker

• ICC

Lab

Immunocytochemistry - Anti-Rab5 antibody - Early Endosome Marker (AB18211)

ab18211 staining Rab5 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab18211 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Project

Western blot - Anti-Rab5 antibody - Early Endosome Marker (AB18211)

All lanes:

Western blot - Anti-Rab5 antibody - Early Endosome Marker (ab18211) at 1 µg/mL

Lane 1:

Western blot - NIH/3T3 whole cell lysate (<a href='/en-us/products/cell-lysates/nih-3t3-whole-cell-lysate-ab7179'>ab7179</a>) at 10 µg

Lane 2:

MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab46770) at 10 µg

Lane 3:

Brain (Mouse) Tissue Lysate (ab27253) at 10 µg

Lane 4:

Testis (Mouse) Tissue Lysate - normal tissue at 10 µg

Lane 5:

Spinal Cord (Mouse) Tissue Lysate at 10 µg

Lane 6:

PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µg

Lane 7:

Brain (Rat) Tissue Lysate - normal tissue at 10 µg

Lane 8:

Heart (Rat) Tissue Lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 24 kDa

Observed band size: 25 kDa

false

• WB

Unknown

Western blot - Anti-Rab5 antibody - Early Endosome Marker (AB18211)

All lanes:

Western blot - Anti-Rab5 antibody - Early Endosome Marker (ab18211) at 1 µg/mL

All lanes:

Western blot - Recombinant Human Rab5A protein (Tag Free) (<a href='/en-us/products/proteins-peptides/recombinant-human-rab5a-protein-ab62956'>ab62956</a>) at 0.001 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 24 kDa

true

Exposure time: 150s

• WB

Project

Western blot - Anti-Rab5 antibody - Early Endosome Marker (AB18211)

All lanes:

Western blot - Anti-Rab5 antibody - Early Endosome Marker (ab18211) at 1 µg/mL

All lanes:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 24 kDa,47 kDa

Observed band size: 24 kDa,40 kDa,67 kDa,70 kDa

false

• WB

CiteAb

Western blot - Anti-Rab5 antibody - Early Endosome Marker (AB18211)

Western Blotting using Anti-Rab5 antibody - Early Endosome Marker, ab18211. Publication image from Zhao, Y. et al., 2020, Nat Commun, 32024826. Legend direct from paper.

Ggpps regulates hepatocyte-derived EV secretion.a EV production by primary hepatocytes from WT and liver-specific Ggpps knockout mice (LKO) fed a normal chow diet or a HFD. (n = 6 mice per group). b CD63, TSG101 and Calnexin protein levels in parental cells and EVs from WT and LKO mice fed a normal chow diet or a HFD. c Membrane-associated Rab27A (lipid-soluble protein with geranylgeranylation in the detergent phase, down) and cytoplasm-associated Rab27A (water-soluble protein with no geranylgeranylation in the aqueous phase, up) were obtained by Triton X-114 extraction and analysed by immunoblotting. (n = 3 biologically independent samples). d Membrane-associated Rab5 (lipid-soluble protein with geranylgeranylation in the detergent phase, down) and cytoplasm-associated Rab5 (water-soluble protein with no geranylgeranylation in the aqueous phase, up) were obtained by Triton X-114 extraction and analysed by immunoblotting. (n = 3 biologically independent samples). e Fluorescence microscopy of primary hepatocytes co-infected with empty vector or Ggpps vector and WT or mutant Rab27A and stained with anti-CD63 antibodies and DAPI (nucleus, blue). (Scale bar, 5 µm). f EV production by primary hepatocytes from e. (n = 6 biologically independent samples). g TSG101 protein levels in the parental cells and EVs from e. Data from 8-week-old WT and LKO mice fed a normal chow diet or a HFD for 12 weeks are presented as the means ± SEM. Groups were analysed using an unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001, WT vs. LKO; ###P < 0.001, Chow vs. HFD. Source data are provided as a Source Data file. See also Supplementary Fig. 5.

false