Overview

  • Product name
    Anti-NCX1 antibody [C2C12]
    See all NCX1 primary antibodies
  • Description
    Mouse monoclonal [C2C12] to NCX1
  • Host species
    Mouse
  • Tested applications
    Suitable for: IHC-P, ICC/IF, IHC-Fr, ELISA, IP, WB, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Guinea pig, Dog, Human, Pig
  • Immunogen

    Full length native protein (purified) corresponding to Dog NCX1. Purified from canine cardiac sodium/calcium exchanger.

  • Epitope
    This antibody recognizes an epitope between amino acids 371-525 on the intracellular side of the plasma membrane.

Properties

Applications

Our Abpromise guarantee covers the use of ab2869 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
IHC-P 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ICC/IF 1/200.
IHC-Fr Use at an assay dependent concentration. PubMed: 21408028
ELISA Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
WB 1/1000. By Western blot, this antibody detects a 120 kDa protein representing the sodium / calcium exchanger from guinea pig cardiac extract. The bands seen at 70 kDa and 160 kDa represent a proteolytic fragment and non-reduced exchanger respectively. This antibody is not recommended for Western blot procedures of rat tissues.
Flow Cyt 1/20 - 1/100.

ab91545 - Mouse monoclonal IgM, is suitable for use as an isotype control with this antibody.

Target

Images

  • ab2869 staining NCX1 in Human kidney tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde, permeabilized with 0.05% Tween20 and blocked with 5% normal goat serum in 1XPBS + 0.05% Tween20 for 1 hour at 25°C; antigen retrieval was by heat mediation in sodium citrate (pH 6.0) buffer. Samples were incubated with primary antibody (1/100 in blocking buffer) for 1 hour at 25°C. Ab47827 (1/500) was used as the secondary antibody.

    See Abreview

  • Overlay histogram showing HEK293 cells stained with ab2869 (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 (ab2869, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgM (mu chain) (ab97007) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgM [ICIGM] (ab91545, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HEK293 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • Immunocytochemistry/Immunofluorescence analysis of NCX1 shows staining in A2058 cells. NCX1 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab2869 (1:100) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of NCX1 shows staining in A549 cells. NCX1 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab2869 (1:100) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

  • Immunocytochemistry/Immunofluorescence analysis of NCX1 shows staining in U251 cells. NCX1 staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were incubated without (control) or with ab2869 (1:100) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

  • This image shows Human embryonic stem cell derived cardiomyocytes, stained with dapi (blue) and anti-NCX1 antibody ab2869 (red). The cells were fixed in paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS and blocked with 4% goat serum for 1 hour. The cells were then incubated with primary antibody (1/100) for 16 hours at 4°C.

    See Abreview

  • Immunohistochemistry was performed on normal deparaffinized Human kidney tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:50 with a mouse monoclonal antibody recognizing Sodium/Calcium Exchanger ab2869 or without primary antibody (negative control; right panel) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry was performed on normal deparaffinized Human heart tissue tissues. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Sodium/Calcium Exchanger ab2869 or without primary antibody (negative control; right panel) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

References

This product has been referenced in:
  • Sahin K  et al. Effects of dietary supplementation of arginine-silicate-inositol complex on absorption and metabolism of calcium of laying hens. PLoS One 13:e0189329 (2018). WB ; Chicken . Read more (PubMed: 29360830) »
  • Cheng PC  et al. Differential regulation of nimodipine-sensitive and -insensitive Ca2+ influx by the Na+/Ca2+ exchanger and mitochondria in the rat suprachiasmatic nucleus neurons. J Biomed Sci 25:44 (2018). Read more (PubMed: 29788971) »
See all 16 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (coronary artery, heart)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate buffer (pH 6)
Permeabilization
No
Specification
coronary artery, heart
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Feb 27 2017

Answer

Thank you and your customerfor taking the time totry the tipsand contact us. I am sorry to hear your customerhas had difficulty obtaining satisfactory results from this antibody.

The details your customerhas kindly provided will provide us with vital information for our monitoring of product quality.

I appreciate the time your customerhas spent in the laboratory and understandtheir concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest your customerhas regrettably received a bad vial.

I apologise for the inconvenience and am pleased to offer your customera free of charge replacement (also possible with an alternative antibody) or credit note in compensation.

In addition, I can suggest your customer may like to consider the following suggestions for future experiments:

Thank you and your customerfor your cooperation. I look forward to hearing from you with details of how your customerwould like to proceed.

Read More

Answer

Thank you and your customer for your inquiry.

I can confirm that ab2869 detects a 120 kDa protein representing the sodium / calcium exchanger from guinea pig cardiac extract. The bands seen at 70 kDa and 160 kDa represent a proteolytic fragment and non-reduced exchanger respectively. The human protein shows the same molecular weight and I assume that bands at 70 kDa and 160 kDa also present a proteolytic fragment and anon-reduced exchanger respectively.

This publications shows the molecular weight of human NCX1:

Liu J et al. Functional sarcoplasmic reticulum for calcium handling of human embryonic stem cell-derived cardiomyocytes: insights for driven maturation. Stem Cells 25:3038-44 (2007).

This publication also uses milk for blocking and a over night incubation of ab2869.

I therefore recommend to following:

1. ) I suggest to use a positive control. Hek293 cells and human kidney are shown to be NCX1 positive.

2.) I can also suggest to check if the secondary antibody works.

3.) NCX1 is a multi pass protein and I therefore do recommend not to boil the samples but only to heat them up to 70C for 10 minutes.

I hope this information is helpful. please do not hesitate to contactme again with any further questions in this matter.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (hESC-derived cardiomyocyte)
Specification
hESC-derived cardiomyocyte
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100 in PBS
Blocking step
Goat serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 04 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Normal Kidney)
Specification
Normal Kidney
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Sodium Citrate pH6.0
Permeabilization
Yes - 0.05% Tween20
Blocking step
Normal Goat Serum in 1X PBS (0.05% Tween20) as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jul 20 2010

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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