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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)

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Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Immunocytochemistry/ Immunofluorescence - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Flow Cytometry (Intracellular) - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Immunoprecipitation - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Flow Cytometry (Intracellular) - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Immunocytochemistry/ Immunofluorescence - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
  • Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR23653-32] to ALIX - BSA and Azide free
  • Suitable for: Flow Cyt (Intra), WB, IP, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Unconjugated

Overview

  • Product name

    Anti-ALIX antibody [EPR23653-32] - BSA and Azide free
    See all ALIX primary antibodies

  • Description

    Rabbit monoclonal [EPR23653-32] to ALIX - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: Flow Cyt (Intra), WB, IP, ICC/IFmore details
    Unsuitable for: IHC-P

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: C6, RAW 264.7, PC-12, NIH/3T3, K-562, HEK-293, HeLa, HCT116, MCF7 and Jurkat whole cell lysates; Human brain tissue lysate; Mouse brain tissue lysate; Rat brain tissue lysate. ICC/IF: NIH/3T3 and HeLa cells. Flow Cyt (intra): NIH/3T3 and HeLa cells. IP: K562 whole cell lysate.

  • General notes

    ab275387 is the carrier-free version of ab275377.

    Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

    This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab275387 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt (Intra)
Use at an assay dependent concentration.
WB
Use at an assay dependent concentration. Detects a band of approximately 80, 90, 100 kDa (predicted molecular weight: 96 kDa).
IP
Use at an assay dependent concentration.
ICC/IF
Use at an assay dependent concentration.
Notes
Flow Cyt (Intra)
Use at an assay dependent concentration.
WB
Use at an assay dependent concentration. Detects a band of approximately 80, 90, 100 kDa (predicted molecular weight: 96 kDa).
IP
Use at an assay dependent concentration.
ICC/IF
Use at an assay dependent concentration.
Application notes
Is unsuitable for IHC-P.

Target

  • Function

    Class E VPS protein involved in concentration and sorting of cargo proteins of the multivesicular body (MVB) for incorporation into intralumenal vesicles (ILVs) that are generated by invagination and scission from the limiting membrane of the endosome. Binds to the phospholipid lysobisphosphatidic acid (LBPA) which is abundant in MVBs internal membranes. The MVB pathway appears to require the sequential function of ESCRT-O, -I,-II and -III complexes. The ESCRT machinery also functions in topologically equivalent membrane fission events, such as the terminal stages of cytokinesis and enveloped virus budding (HIV-1 and other lentiviruses). Appears to be an adapter for a subset of ESCRT-III proteins, such as CHMP4, to function at distinct membranes. Required for completion of cytokinesis. Involved in HIV-1 virus budding. Can replace TSG101 it its role of supporting HIV-1 release; this function implies the interaction with CHMP4B. May play a role in the regulation of both apoptosis and cell proliferation.

  • Sequence similarities

    Contains 1 BRO1 domain.

  • Cellular localization

    Cytoplasm > cytosol. Melanosome. Cytoplasm > cytoskeleton > centrosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. Colocalized with CEP55 in the midbody during cytokinesis. Colocalized with CEP55 at centrosomes of non-dividing cells.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • AIP1 antibody
    • ALG 2 interacting protein 1 antibody
    • ALG-2-interacting protein 1 antibody
    • ALG2 interacting protein X antibody
    • Alix antibody
    • Apoptosis linked gene 2 interacting protein X antibody
    • Dopamine receptor interacting protein 4 antibody
    • DRIP4 antibody
    • Hp95 antibody
    • KIAA1375 antibody
    • MGC17003 antibody
    • PDC6I_HUMAN antibody
    • PDCD6 interacting protein antibody
    • PDCD6-interacting protein antibody
    • PDCD6IP antibody
    • Programmed cell death 6 interacting protein antibody
    • Programmed cell death 6-interacting protein antibody
    see all

Images

  • Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    All lanes : Anti-ALIX antibody [EPR23653-32] (ab275377) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293 cell lysate
    Lane 2 : PDCD6IP knockout HEK-293 cell lysate
    Lane 3 : HeLa cell lysate
    Lane 4 : Human Spleen tissue lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 96 kDa
    Observed band size: 96 kDa



    This data was developed using the same antibody clone in a different buffer formulation (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 ALIX 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.

  • Immunocytochemistry/ Immunofluorescence - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Immunocytochemistry/ Immunofluorescence - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)

    This data was developed using ab275377, the same antibody clone in a different buffer formulation.

    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.

  • Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    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: 100,80,90 kDa why is the actual band size different from the predicted?



    This data was developed using ab275377, the same antibody clone in a different buffer formulation. 

    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 Cytometry (Intracellular) - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Flow Cytometry (Intracellular) - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)

    This data was developed using ab275377, the same antibody clone in a different buffer formulation. 

    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) (Black) isotype control 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. 

     

     

  • Immunoprecipitation - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Immunoprecipitation - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)

    This data was developed using ab275377, the same antibody clone in a different buffer formulation.

    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

  • Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    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: 100,80,90 kDa why is the actual band size different from the predicted?



    This data was developed using ab275377, the same antibody clone in a different buffer formulation. 

    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 Cytometry (Intracellular) - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Flow Cytometry (Intracellular) - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)

    This data was developed using ab275377, the same antibody clone in a different buffer formulation. 

    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. 

     

     

  • Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Western blot - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    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?



    This data was developed using ab275377, the same antibody clone in a different buffer formulation. 

    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.

  • Immunocytochemistry/ Immunofluorescence - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Immunocytochemistry/ Immunofluorescence - Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)

    This data was developed using ab275377, the same antibody clone in a different buffer formulation.

    Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic 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.

  • Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)
    Anti-ALIX antibody [EPR23653-32] - BSA and Azide free (ab275387)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (0)

Publishing research using ab275387? Please let us know so that we can cite the reference in this datasheet.

ab275387 has not yet been referenced specifically in any publications.

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