Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling ALIX with ab275377 at 1/5000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488, ab150077) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HeLa cells is observed. Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594, ab195889) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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

All lanes : Anti-ALIX antibody [EPR23653-32] (ab275377) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Rat brain tissue lysate
Lane 3 : C6 (rat glial tumor glial cell) whole cell lysate
Lane 4 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 6 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 96 kDa
Observed band size: 80/90/100 kDa why is the actual band size different from the predicted?

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

The expression pattern is consistent with what has been described in the literature (PMID: 24834918, 26935291, 28322231).

Exposure time: Lane1-2: 10 seconds Lane3-6: 8 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell line) cells labelling ALIX with ab275377 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730)  isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

ALIX was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast cell line) whole cell lysate with ab275377 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab275377 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: K-562 whole cell lysate 10 ug

Lane 2: ab275377 IP in K-562 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab275377 in K-562 whole cell lysate

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

Exposure time: 3 seconds

Anti-ALIX antibody [EPR23653-32] (ab275377) at 1/1000 dilution + Human brain tissue lysate at 20 µg

Secondary
VeriBlot for IP secondary antibody(HRP)(ab131366) at 1/1000 dilution

Predicted band size: 96 kDa
Observed band size: 80/90/100 kDa why is the actual band size different from the predicted?

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

The expression pattern is consistent with what has been described in the literature (PMID: 24834918, 26935291, 28322231).

Exposure time: 10 seconds.

 

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast cell line) cells labelling ALIX with ab275377 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-ALIX antibody [EPR23653-32] (ab275377) at 1/1000 dilution

Lane 1 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 2 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 5 : MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 6 : Jurkat (human T cell leukemia T lymphocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 96 kDa
Observed band size: 90/100 kDa why is the actual band size different from the predicted?

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

The expression pattern is consistent with what has been described in the literature (PMID: 24834918, 26935291, 28322231).

Exposure time: 10 seconds.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embyro fibroblast cell line) cells labelling ALIX with ab275377 at 1/5000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488, ab150077 ) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cells. Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594, ab195889) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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