Anti-Nodal antibody [5C3] (ab55676)

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

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.

Thank you for taking the time to respond and for contacting Abcam.

With over 70,000 products it is logistically impossible for us to offer free samples. I hope you understand.

Please let me know if you have any questions.

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.

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.

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.