FITC-Rabbit IgG  - Isotype Control (ab37406)

Thank you very much for contacting us with your question, and for your patience.
I have contacted the lab scientists, and the F:P ratio is between 3 and 6 for this antibody.
I hope this information is useful, but please let me know if you have further questions or if there is anything else that we can do for you.

We have done stability studies by storing conjugated antibodies at different temperature. The study shows FITC conjugated products are more stable at 4C rather than -20C so in response to this we changed the datasheets of all conjugated antibody we have in catalogue.
FITC conjugate will be stable for 2 years at 4C.
Please note the Abpromise guarantee period is 1 year from date of purchase.

Thank you for contacting us and your interest in our products.

The Rabbit IgG FITC - Isotype Control (ab37406) of the lot number GR59579-1 is provided at a concentration of 0.1mg/mL and is supplied as 1 mL.

As the antibody is intended to be used as an isotype control we would advise using the antibody with the same protocol you intend to use with the rabbit IgG antibody you are using to detect a target in your experiment.

I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

Thank you for your inquiry.
I am happy to confirm that ab37406 (lot GR77642) has a concentration of 0.1mg/ml.
I hope this information is helpful and wish you good luck with your research.

Thank you for your clarification.

I can confirm the concentration of ab66219 (Batch: GR9812) of this ab is 1 mg/ml. The other antibody ab36204 (Lot: GR42382) is not affinity purified but whole antiserum so the IgG is not quantified. However, I can provide a range of the IgG: 10-15 mg/ml.

Isotype controls:



can be searched at this site: https://www.abcam.com/index.html?c=718

ab36204, ab48614, ab66219, ab102998 --> Rabbit IgG - ab37406, ab125938, ab128142

ab83508 --> Mouse IgG1 - ab91353, ab27479, ab81032



Positive control:



ab36204 - CRMP4 - It is mainly expressed in heart and skeletal muscle and also strongly expressed in fetal brain and spinal cord (human, mouse or rat),

ab48614 --Ceruloplasmin - It is expressed by the liver and secreted in plasma,

ab66219 -- Oprl1 - Brain (cortex),

ab83508 -- NG2 - It is detected only in malignant melanoma cells, SK-MEL-28 cells,

ab102998 --B3GNT5- only tested in WB - Widely expressed. It is highly expressed in lung, colon, placenta, testis, pituitary gland and cerebellum, MDA-MB435 cell line

If you need any further assistance in the future, please do not hesitate to contact me.

I think its good that you are checking the cell viability and using HeLa may provide more information as to if it is the cell strain or the antibodies that may be contributing to the problems. I agree that checking both the primary and secondary antibody is excessive but if you have access to an alternative secondary antibody it may be worthwhile trying. The activity of the secondary antibody should not have been compromised as you are storing it as we have recommended (and I have checked that these guidelines are correct). The antibody should also be stored in the dark, as should ab37406. Ab37406 and ab83174 should be aliquotted (we usually recommend no lower than 10 uL) and stored at -20 or -80. After thawing an aliquot this should not be refrozen but can be storedat 4degreesfor a few weeks if some is left over after your experiment.


Let me know how you get on with your experiments. I'll be crossing my fingers for you.

Thank you for providing such a full explanation of what you are trying to achieve. I can now understand exactly why you have chosen the technique that you have.

I think the experiments you are planning to do next will be very useful in determining how best to proceed with the staining and more fully understandingwhere the non-specificity iscoming from.I too hope they will be brilliant! I don't knowif you areplanning on it yet but I would suggest keeping at least oneof your test withoutair dryingyourcells(i.e keeping them hydrated throughout), just to seeif thisimproves the non-specificity seen with the secondary and isotype control antibodies. If it does, it would make youractual experimentdifficult to measure theIR butI think it is most important at the moment toget the specific staining. Once this has been achieved, the optimisationof how toperform the actual experiments can be done.

I wish you all the best with the new experiments and look forward to hearing how you get on.

Thank you for getting back to me with your new results. It is good that we can now say conclusively that there is non-specific binding of both the secondary antibody and of the isotype control antibody.

Following the protocol changes, I do not exactly know why this non-specific nuclear staining is persisting but I have a few suggestions to add that may be beneficial.

I am not an expert in using cytospin by any means so I have been having a read about it. It seems that it is generally recommended to first spin your suspension, then fix the cells on the slides. The following reference may be of help to you:

http://www.ihcworld.com/_protocols/histology/cytospin.htm

http://sciencepark.mdanderson.org/labs/tang/cytospin4web.html

It may also be beneficial to fix immediately, then stain immediately. I would not allow your cellsto dry out at any stage.The only Abreview we have of ab83174 having been used in ICC used PFA fixation and some non-specific staining of the cytoplasm was also seen (which seems to be what you are seeing). We have charaterised this target using methanol fixation (refer toab83174 datasheet). It maybe worthwhile to try this in order to improve the staining.

Is there a reason why you are using cytospin? If your cells are adherent you could just grow the cells on glass slides directly as is described in the following protocol:

https://www.abcam.com/index.html?pageconfig=resource&rid=11417

I have a further question. What diluent are you using for your antibodies? I would suggest still usingthe same asyouwere (1% BSAin PBST).Are you performingany washing steps? Following each incubation I would wash theslide with PBS 3 times for 5 minutes.

If you have any questions in regards to my suggestions please do let me know.

Thank you for that last bit of information.


I just wanted to check that the isotype control wasn't being used in a higher concentration than the primary antibody, but everything is fine.


I look forward to hearing how you get on.

Sorry for the delay in getting back to you and thank you for all that information. It makes it much easier to understand what is going on and what has been tried previously.

I think the next step you are suggestingare good. The blocking buffer and antibody diluent you are using are both what were used in performing inhouse testing with this antibody. Additionally, performing the following controls:

1. isotype control only

2. secondary antibody only (no-primary control)

3. primary antibody+secondary antibody

you will see more accurately what element is contributing to the non-specific signal.

There are a few things I would add though.

1. for fixation with formalin we would normaly say 10-15 minutes would be enough. The danger in prolonging the fixation is that the epitope that the antibody recognises can be masked, requiring antigen retrieval to be employed. It can also lead to increased non-specific binding. I would therefore suggest reducing the fixation time.

2. the recommended dilution for the secondary antibody you are using is 1/1000 to 1/2000. This is equivalent to 1-2 µg/mL. I would therefore reduce the concentration used from 4 µg/mL to 1 µg/mL, which will hopefully help in reducing the non-specific signals observed.

Additionally, may I ask how thedilutionof the isotype control was performed?

The isotype that you are using I would not specifically recommend as a control for the experiment you are performing. As the antibody is already FITC conjugated, if it does bind non-specifically and the secondary also binds, you will get an amplification of the signal over and above what the actual primary antibody non-specificity would yield. I would either suggest using a primary antibody that is directly FITC conjugated, or an isotype control such as ab27478.

Ihope the suggestions I have made improve the results seen so far. I look forward to hearing how you get on.