Overview

  • Product name

  • Description

    Mouse monoclonal to Nodal
  • Host species

    Mouse
  • Tested applications

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

    Reacts with: Human
  • Immunogen

    Recombinant fragment: RCEGECPNPV GEEFHPTNHA YIQSLLKRYQ PHRVPSTCCA PVKTKPLSML YVDNGRVLLD HHKDMIVEEC GC, corresponding to amino acids 275-347 of Human Nodal

  • General notes

    This product was changed from ascites to tissue culture supernatant on 28th May 2019. Please note that the dilutions may need to be adjusted accordingly. If you have any questions, please do not hesitate to contact our scientific support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab55676 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. Predicted molecular weight: 40 kDa.
IHC-P Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

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

 

Target

  • Function

    Essential for mesoderm formation and axial patterning during embryonic development.
  • Involvement in disease

    Defects in NODAL are the cause of visceral heterotaxy autosomal type 5 (HTX5) [MIM:270100]. A form of visceral heterotaxy, a complex disorder due to disruption of the normal left-right asymmetry of the thoracoabdominal organs. It results in an abnormal arrangement of visceral organs, and a wide variety of congenital defects. Clinical features of visceral heterotaxy autosomal type 5 include situs inversus viscerum or situs ambiguus, congenital heart defect, transposition of the great vessels ventricular septal defect, atrial septal defect, truncuscommunis, and dextrocardia.
  • Sequence similarities

    Belongs to the TGF-beta family.
  • Cellular localization

    Secreted.
  • Information by UniProt
  • Database links

  • Alternative names

    • HTX5 antibody
    • MGC138230 antibody
    • Nodal antibody
    • Nodal Growth Differentiation Factor antibody
    • nodal homolog (mouse) antibody
    • Nodal homolog antibody
    • Nodal, Mouse, Homolog antibody
    • NODAL_HUMAN antibody
    see all

Images

  • Nodal antibody (ab55676) used in immunohistochemistry at 3ug/ml on formalin fixed and paraffin embedded human endometrium cancer.

    This image was generated using the ascites version of the product.

  • Nodal antibody (ab55676) at 1ug/lane + HeLa cell lysate at 25ug/lane.

    This image was generated using the ascites version of the product.

  • ICC/IF image of ab55676 stained mouse embryonic stem cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab55676, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

    This image was generated using the ascites version of the product.

  • Overlay histogram showing HEK293 cells stained with ab55676 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab55676, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. 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 HEK293 cells fixed with 100% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

    This image was generated using the ascites version of the product.

References

This product has been referenced in:

See all 12 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Answer

Thank you for your reply.

I am sorry about the confusion whether ab55676 was tested in IF or not, as you say the image on the datasheet clearly shows that is does work in IF, I am sorry I missed that.

Having, looked over your protocol, I am in agreement with you that the issue seems to be non-specific binding of your secondary antibody. The only part of the protocol that I would say to be careful about is your block, as you should match the block to the host species of your secondary. I only mention this as you say you use goat serum for blocking but one of the secondary’s you used was raised in donkey. Also you do not need to incubate secondary antibodies overnight at 4C, in most cases 60 minutes at room temp is more than sufficient and any longer can cause high background.

Therefore before you purchase a different secondary antibody, I would recommend that you repeat the experiment as before but alter the incubation time of your secondary antibody.

Please let me know if there is anything else I can help you with

Read More
Application
Western blot
Sample
Horse Tissue lysate - whole (Endometrium)
Gel Running Conditions
Reduced Denaturing (6% Stacking Gel, 10% Resolving Gel)
Loading amount
6 µg
Specification
Endometrium
Blocking step
BSA in PBS+Tween-20 as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Apr 12 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (1 week Retinoic acid differentiation of NTERA-2)
Specification
1 week Retinoic acid differentiation of NTERA-2
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Oct 21 2015

Application
Western blot
Loading amount
60 µg
Gel Running Conditions
Reduced Non-Denaturing (Native) (12)
Sample
Human Cell lysate - whole cell (LNCaP cell line)
Specification
LNCaP cell line
Blocking step
Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C

Dr. Francesco Elia Marino

Verified customer

Submitted May 15 2014

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
CAS BLOCK as blocking agent for 20 minute(s) · Concentration: 100% · Temperature: 26°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Sodium Citrate Buffer
Sample
Human Tissue sections (Human Prostate)
Specification
Human Prostate
Permeabilization
No
Fixative
Paraformaldehyde

Dr. Francesco Elia Marino

Verified customer

Submitted May 15 2014

Answer

Thank you for your reply.

I am sorry that I misread your original email with the protocol information, I know see that you tried the secondary antibody with both 15 and 30 minutes incubation times but that did not make a difference.

Below are links to antibodies that I believe will work well for you, there are 3, all with a different fluorescent tag on them so that you can pick one based on which color you want to use:

https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-DyLight-488-pre-adsorbed-ab96899.html
https://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-DyLight-650-pre-adsorbed-ab96898.html
https://www.abcam.com/Donkey-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-DyLight-594-pre-adsorbed-ab98500.html



Under our Abpromise, all of the above antibodies are guaranteed to work in IF and if for whatever reasons the antibody fails to work as stated then we will be happy to either to replace or refund the antibody.

If there is anything else I can help you with, please let me know.

Read More

Answer

Thank you for contacting Abcam.

I am sorry that you are having problems with ab55676 in immunofluorescence. As we had not tested this antibody in IF before, we were not sure how the antibody would work in that application. I would be very happy to see if I can give any protocol suggestions that would improve the staining that you are getting.

Could you provide me with a description of the protocol that you are using, sample types, type of fix, blocking agent used, primary antibody concentration and incubations times. Also do you use any type of detergent to permeablilze your samples.

I look forward to your reply and helping you to achieve better staining.

Read More

Answer

Thank you for contacting Abcam.

I am sorry to hear that Ab55676 has not performed well for you. Although the peptide sequence has excellent identity with the mouse homolog, this product is not guaranteed to work in mouse tissues. High background is a very common issue when working in tissues of the same species as the host speciesof the primary anti-body. These may be reduced by using a secondary antibody that has been pre-absorbed or by direct conjugation of your label to the primary antibody.

Our EasyLink conjugation kits offer an effective and simple way to directly conjugate labels to your primary antibody. More information may be found at the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=13148&source=pagetrap&viapagetrap=easylink

We have 12 secondary antibodies against mouse IgG1, they may be viewed at the following link:

https://www.abcam.com/index.html?pageconfig=searchresults&search=IgG1&pt=7&sk=targsp&sv=2&sn=Mouse&l=2&fViewMore=1

Please let me know if you have any questions.

Read More

Answer

Further to my previous response to your complaint, I would like to pass some more information which you may find useful. The antibody is (NODAL monoclonal antibody (ab55676) has been tested and characterized in Western blot on several cell lines, such as HeLa, PC-12, and NIH/3T3. I would suggest using one of these cell lines (if you have access to them) as good well characterized positive control alongside with the samples. Please find the cell lysate preparation (denatured) protocol below: Step-by-step procedure: 1. Prepare cell lysate by homogenization in ice-cold modified RIPA Lysis Buffer. And add sufficient volume of protease Inhibitor (add 100 ul every 1mL RIPA Lysis Buffer), then vortex. 2. After centrifugation, determine the protein concentration of the cell lysate. 3. Boil the lysate for 10 minutes in 1X SDS sample buffer. 4. The lysate is ready to load onto SDS-PAGE for Western blotting. (20~50 μg per lane is recommended for mini gel.) Tested Applications: Western blot Reagents: _ RIPA Lysis Buffer: 50mM Tris-HCl pH 7.4,150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1mM PMSF( PMSF should be added before use) _ 1X SDS sample buffer: 2% SDS, 300mM, 300mM _-mercaptoethanol, 50mM Tris-HCl pH 6.8,10% Glycerol, 0.01% Bromophenol blue If you need any further assistance in the future, please do not hesitate to contact me.

Read More

1-10 of 12 Abreviews or Q&A

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