Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-GSK3 beta + GSK3 alpha antibody [EPR18814-102] - BSA and Azide free (ab226169)

Overview

  • Product name

    Anti-GSK3 beta + GSK3 alpha antibody [EPR18814-102] - BSA and Azide free
  • Description

    Rabbit monoclonal [EPR18814-102] to GSK3 beta + GSK3 alpha - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, ICC/IF, Flow Cyt, IPmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Recombinant fragment within Mouse GSK3 beta aa 200 to the C-terminus. The exact sequence is proprietary.
    Database link: Q9WV60

  • Positive control

    • IHC-P: Mouse testis tissue.
  • General notes

    Ab226169 is the carrier-free version of ab185141. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    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.

    ab226169 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

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

    This product is a recombinant rabbit monoclonal antibody.

Applications

Our Abpromise guarantee covers the use of ab226169 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
WB Use at an assay dependent concentration. Detects a band of approximately 47, 52 kDa (predicted molecular weight: 47, 52 kDa).
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This antibody is not recommended for IHC in human.

ICC/IF Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.
IP Use at an assay dependent concentration.

Target

Images

  • All lanes : Anti-GSK3 beta + GSK3 alpha antibody [EPR18814-102] (ab185141) at 1/5000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : GSK3 alpha knockout HAP1 whole cell lysate
    Lane 3 : GSK3 beta whole cell lysate
    Lane 4 : HeLa whole cell lysate
    Lane 5 : Hek293 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 47, 52 kDa



    Lanes 1 - 5: Merged signal (red and green). Green - ab185141 observed at 47/52 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab185141 was shown to specifically react with GSK3 alpha and GSK3 beta in wild-type HAP1 cells as signal was lost in GSK3 alpha and GSK3 beta knockout cells. Wild-type and GSK3 alpha and GSK3 beta knockout samples were subjected to SDS-PAGE. ab185141 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/10000 dilution for 1 hour at room temperature before imaging.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

  • Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling GSK3 beta + GSK3 alpha with ab185141 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on rat testis (PMID: 22792253). Counter stained with hematoxylin.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling GSK3 beta + GSK3 alpha with ab185141 at 1/150 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cells.

    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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

  • Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling GSK3 beta + GSK3 alpha with ab185141 at 1/150 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cells.

    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.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

  • Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cell line labeling GSK3 beta + GSK3 alpha with ab185141 at 1/60 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 H&L (Alexa Fluor® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

  • GSK3 beta + GSK3 alpha was immunoprecipitated from 0.35 mg of NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate with ab185141 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab185141 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

    Lane 1: NIH/3T3 whole cell lysate 10 µg (Input). 

    Lane 2: ab185141 IP in NIH/3T3 whole cell lysate. 

    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab185141 in NIH/3T3 whole cell lysate.

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

    Exposure time: 3 seconds.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

  • Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling GSK3 beta + GSK3 alpha with ab185141 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining on mouse testis (PMID: 22792253). Counter stained with hematoxylin.

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

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab185141).

    Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

References

ab226169 has not yet been referenced specifically in any publications.

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