Anti-ALIX antibody [EPR23653-32]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ALIX antibody [EPR23653-32] (AB275377)

Intracellular 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ALIX antibody [EPR23653-32] (AB275377)

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.

• IP

Unknown

Immunoprecipitation - Anti-ALIX antibody [EPR23653-32] (AB275377)

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

All lanes:

Immunoprecipitation - Anti-ALIX antibody [EPR23653-32] (ab275377)

Predicted band size: 96 kDa

Observed band size: 90-100 kDa

false

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-ALIX antibody [EPR23653-32] (AB275377)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-ALIX antibody [EPR23653-32] (AB275377)

Intracellular 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.

• WB

Lab

Western blot - Anti-ALIX antibody [EPR23653-32] (AB275377)

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.

All lanes:

Western blot - Anti-ALIX antibody [EPR23653-32] (ab275377) at 1/1000 dilution

All lanes:

Human brain tissue lysate at 20 µg

Secondary

All lanes:

VeriBlot for IP secondary antibody(HRP)(<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 96 kDa

Observed band size: 100 kDa,80 kDa,90 kDa

false

• WB

Lab

Western blot - Anti-ALIX antibody [EPR23653-32] (AB275377)

Lanes 1 - 4 : Merged signal (red and green). Green - ab275377 observed at 96 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab275377 was shown to react with PDCD6IP in wild-type HEK-293T cells in Western blot with loss of signal observed in PDCD6IP knockout cell line ab260864 (PDCD6IP knockout cell lysate ab261656). Wild-type HEK-293T and PDCD6IP knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5 % milk in TBS-T (0.1 % Tween^®) before incubation with ab275377 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-ALIX antibody [EPR23653-32] (ab275377) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 cell lysate at 20 µg

Lane 2:

PDCD6IP knockout HEK-293 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Human Spleen tissue lysate at 20 µg

Predicted band size: 96 kDa

Observed band size: 96 kDa

false

• WB

Lab

Western blot - Anti-ALIX antibody [EPR23653-32] (AB275377)

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.

All lanes:

Western blot - Anti-ALIX antibody [EPR23653-32] (ab275377) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HCT116 (human colorectal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 96 kDa

Observed band size: 90-100 kDa

false

• WB

Lab

Western blot - Anti-ALIX antibody [EPR23653-32] (AB275377)

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.

All lanes:

Western blot - Anti-ALIX antibody [EPR23653-32] (ab275377) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Lane 3:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 4:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 5:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate at 20 µg

Lane 6:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 96 kDa

Observed band size: 100 kDa,80 kDa,90 kDa

false