All lanes : Anti-Rab5 antibody [EPR21801] (ab218624) at 1/1000 dilution

Lane 1 : Wild type HAP1 whole cell lysate
Lane 2 : Rab5 knockout HAP1 whole cell lysate
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

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


Exposure time: 15 seconds

ab218624 was shown to specifically react with Rab5 in wild-type HAP1 cells as signal was lost in Rab5 knockout cells. Wild-type and Rab5 knockout samples were subjected to SDS-PAGE. Ab218624 and ab181602 (Human anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/200000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD® ChemiDoc™ MP instrumentusing the ECL technique.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed wild-type and RAB5A KO HAP1 cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing no staining in RAB5A KO HAP1 cell line and granular cytoplasmic staining in parental HAP1 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes : Anti-Rab5 antibody [EPR21801] (ab218624) at 1/1000 dilution

Lane 1 : Neuro-2a (mouse neuroblastoma cell line) whole cell lysate
Lane 2 : Human fetal brain lysate
Lane 3 : Human fetal heart lysate
Lane 4 : Human fetal spleen lysate
Lane 5 : Rat brain lysate
Lane 6 : Rat heart lysate
Lane 7 : Mouse brain lysate
Lane 8 : Mouse heart lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

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

 

Exposure time :  Lane 1: 5 seconds; Lanes 2/8: 15 seconds; Lanes 3/4: 6 seconds; Lanes 5/7: 3 seconds;  Lane 6: 26 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in human kidney is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in human cerebrum is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in mouse kidney is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Rab5 with ab218624 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in rat liver is observed (PMID 7789520). Counterstained with hematoxylin. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed NIH/3T3 (mouse embryo fibroblast cell line) cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing granular cytoplasmic staining in NIH/3T3 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilzed  HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Rab5 with ab218624 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing granular cytoplasmic staining in HeLa cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized MCF7 (human breast adenocarcinoma cell line) cell line labelling Rab5 with ab218624 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Rab5 was immunoprecipitated from 0.35 mg MCF7 (human breast adenocarcinoma cell line) whole cell lysate with ab218624 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab218624 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1: MCF7 whole cell lysate 10 μg (input).

Lane 2: ab218624 IP in MCF7 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab218624 in MCF7 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second.