Overview

  • Product name

    Anti-CNPase antibody [11-5B] - BSA and Azide free
    See all CNPase primary antibodies
  • Description

    Mouse monoclonal [11-5B] to CNPase - BSA and Azide free
  • Host species

    Mouse
  • Specificity

    Reacts specifically with CNPase. In an immunoblotting assay, the antibody localizes both CNP1 (46kD) and CNP2 (48kD) bands. Immunohistochemical staining of paraffin, cryostat or vibratome sections of rat brain reveals selective staining of oligodendrocytes in the grey and white matter. Nerve cells and axons are not stained and astroglial cells do not appear to be labelled.

  • Tested applications

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

    Reacts with: Mouse, Rat, Human
    Does not react with: Chicken, Guinea pig
  • Immunogen

    Full length native protein (purified) corresponding to Human CNPase.

  • Positive control

    • IHC-P: FFPE human cerebral cortex tissue sections; ICC/IF: SKNSH cell line; Flow: SH-SY5Y cells; WB: Human spinal cord and brain lysates, Mouse spinal cord and brain lysates, Rat brain tissue lysates.
  • General notes

    Ab237961 is a PBS-only buffer format of ab6319. Please refer to ab6319 for recommended dilutions, protocols, and image data.

     

Properties

Applications

Our Abpromise guarantee covers the use of ab237961 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 a concentration of 5 µg/ml. Predicted molecular weight: 48 kDa.
ICC/IF Use a concentration of 10 µg/ml.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

IHC-P 1/1000 - 1/2000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

Images

  • ab6319 stained in SKNSH cells. Cells were fixed with 100% methanol (10 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% Triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab6319 at 10µg/ml and ab6046 (Rabbit polyclonal to beta Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab6319).

  • Lane 1: Wild-type HAP1 whole cell lysate (40 µg)
    Lane 2: CNPase knockout HAP1 whole cell lysate (40 µg)
    Lane 3: Human Brain whole cell lysate (20 µg)

    Lanes 1 - 3: Merged signal (red and green). Green - ab6319 observed at 48 kDa. Red - loading control, ab181602, observed at 37 kDa.

    ab6319 was shown to recognize CNPase in wild-type HAP1 cells as signal was lost at the expected MW in CNPase knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CNPase knockout samples were subjected to SDS-PAGE. Ab6319 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 5 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 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, L-Arginine and sodium azide (ab6319).

  • IHC image of CNPase staining in human cerebral cortex formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab6319, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab6319).

  • Overlay histogram showing SH-SY5Y cells stained with ab6319 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab6319, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab6319).

  • All lanes : Anti-CNPase antibody [11-5B] (ab6319) at 1/100 dilution

    Lane 1 : ab29188
    Lane 2 : ab29466
    Lane 3 : Spinal Cord (Mouse) Tissue Lysate
    Lane 4 : Brain (Mouse) Tissue Lysate
    Lane 5 : Spinal Cord (Rat) Tissue Lysate
    Lane 6 : Brain (Rat) Tissue Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Predicted band size: 48 kDa



    This antibody was raised against full length native CNPase and is predicted to recognize both isoforms. The predicted molecular weights of isoforms CNPI and CNPII are 45- and 48-kDa respectively.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, L-Arginine and sodium azide (ab6319).

References

ab237961 has not yet been referenced specifically in any publications.

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