Recombinant Anti-Adenosyl Homocysteine antibody [EPR4499] (ab111903)

The competitive ELISA protocol is as follow:

1. 0.1 ug/ml of BSA-S-adenosylhomocysteine was coated into 96 wells.50ul/well,4 ℃，overnight.

2. Add 120µl/well of 1% BSA-TBS and incubate for 1 hours at RT.

3.Wash the plates one time with 1× TBST, 200 ull/well.

4. Add 25ul Serial dilution of S-adenosylhomocysteine (SAH), SAMe, et al.and 25ul 0.05 ug/ml of ab111903 to 96 wells and incubate for 1hours in shaker at 37⁰C

5.Wash the plates 3 times with 1× TBST, 200 ull/well

6.Add 50ul/well HRP conjugated Goat anti-rabbit IgG into ELISA Plate , 40mins.

7.Add 50ul/well TMB

8.Add 50ull of 1M H2SO4 when signal reaches optimal level.

Thank you for your enquiry.

I can confirm that ab111903 antibody is sold as tissue culture supernatant. Unpurified antibodies, such as those sold as whole antiserum, ascites or tissue culture supernatant will not have a concentration stated on the datasheet. Antibody concentration is usually determined by protein assay, and tissue culture supernatant will contain a lot of other proteins, which means the antibody quantification would not be accurate.

I can confirm that for tissue culture supernatant, concentration of antibody is known to vary between 1 - 3 mg/ml

I am sorry we are not able to provide an exact concentration on this occasion, but hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

I meant to add that if you would be able to explain a little more fully what it is you are studying and what you are trying to achieve, I may be able to help you with other ideas of how to achieve it.
I look forward to your reply.

I have had a chat to my colleague who is very experienced with flow cytometry in regards to how to optimise these antibodies for this application.
Unfortunately, neither of these antibodies have been used for this application previously and optimisation would be required. As the SAH is located in the cytoplasm, permeabilization will be required to allow the antibody to reach the target. This would also potentially allow SAH to move out of the cell. Fixation of the cell is therefore necessary, but this could potentially cause problems in the detection of the molecule as the epitope may be disguised. In addition to this, as the antibodies are not directly conjugated, a secondary antibody would be required and therefore an additional incubation and washing step. This can be problematic and increase the non-specificity of the experiment. An alternative would be to directly conjugate the ab35164, (for example with one of your EasyLink kits) however this adds expense and time to what you are doing. It may be marginally easier to perform ICC experiments with your cells as this would be more amenable to having a secondary antibody and you would be able to see more easily if non-specific binding is occurring. However, if you are not seeing strong binding to the molecule in an ELISA format, I would suggest moving to a flow system or ICC is unlikely to improve this situation.
We are unfortunately unable to invite you into our labs to optimise the ELISA conditions. Both of these antibodies are sourced (and validated) from collaborators and as such I cannot offer this oppertunity. How are you finding the ELISA with the ab35164? You said that this had improved? Are you seeing a specific signal? Is it being competed away with addition of the SAH?
I am sorry that I could not be of more help. I look forward to receiving your reply.

Thank you for your enquiry.

I can confirm that Anti-Adenosyl Homocysteine antibody (ab111903) is sold as tissue culture supernatant. Unpurified antibodies, such as those sold as whole antiserum, ascites or tissue culture supernatant will not have a concentration stated on the datasheet. Antibody concentration is usually determined by protein assay, and tissue culture supernatant will contain a lot of other proteins, which means the antibody quantification would not be accurate.For tissue culture supernatant, the concentration of antibody usually to varies between 1 - 3 mg/ml.

As this product is Rabbit IgG, purification is possible with a protein G affinity column.

I am sorry we are not able to provide an exact concentration on this occasion, but hope this information will be helpful to you. If you have any further questions, please do not hesitate to contact us.

Thank you for providing that extra information. It is unfortunate that the ab111903 has not out-performed ab35164 so far.

Do you see any signal at all with ab111903? The only thing I can suggest having looked at your protocol and taking into consideration that ab35164 is showing a signal is that ab111903 may not perform at its best with incubation at 37 degrees. The antibody was validated with incubation at room temperature for 1 hour. It may therefore be worthwhile to see if the signal improves with incubation under these conditions and maybe also trying 4 degrees, overnight.

What I am also checking is how the immunogen used for ab111903 was made, i.e. how the SAH was conjugated to the carrier protein, just to check that the epitope that the antibody is likely to detect would be present in your sample.

Another comment, as SAH is liable to oxidation, have you introduced any protective agent when performing the blocking step?

Sorry for the delay in getting back to you.


I am very sorry to hear that the ab111903 has not performed as well as ab35164. In order to try and understand what is going on would you please be able to send me some protocol details again. I think you have gone through so much optimisation that the original information I have from when we first started to correspond must be very out of date by now. I have attached a questionnaire for this purpose. If you could also send me the raw data you have for the new experiments (both with ab35164 and ab111903) would be very useful. I'll then try to consult with some additional people in the lab to get their opinion on this again.


I am very sorry that you have had such a lot of trouble with these antibodies. I'll try my best to help you find out why this experiment has not worked.


I look forward to receiving your reply.

I'm glad you have seen improvements in using ab35164. May I ask, what do you think has lead to these improvements? It would be very useful information to know in order to advise customers in the future.


Unfortunately due to the size of our catalogue we are unable logistically to provide sample sizes of products. However, as a good-will gesture to acknowledge all the work you have put in optimising the protocol with this antibody I can offer you a vial of ab111903free of charge.


If I send this to you would you please fill out Abreviews for both ab35164 and ab111903? This should only take a few minutes for each antibody and would be invaluable information for future customers to use in choosing the right product for them. This will also get you Abpoints which can be used to redeem money off Abcam products or Amazon vouchers.


More information on the Abreview system can be found from the following link:


https://www.abcam.com/abreviews


I look forward to hearing how you would like to proceed.

Thank you for your enquiry.

You are correct that there is moderate cross-reactivity to SAMe. I'd put this in the range of 1-4%. I apologize for the error and will have the datasheet updated.

I hope this is helpful. Please contact me again if you have any further questions.

Thank you for the extra details you provided. In answer to your questions:

1. Could you possibly tell which supplier of SAH you used for standards when testing your antibody, as we have discovered that it may be susceptible to oxidation in both liquid and solid forms.

A: The standards were mainly purchased from Sigma Aldrich. For the Adenosyl-Homocyteine, we used Sigmacatalogue number A9384. This was then conjugated to BSA to facilitate coating on to plates.

2. Could you possibly tell me how you reconstituted the SAH and at what concentrations you used it at to generate your standards?

A: The standards were prepared in TBS containing 0.1% Tween 20, pH 8.0. We used the following working dilutions:

2mg/ml, 20 ug/ml, 200 ng/ml, 2 ng/ml, 2 pg/ml

I hope this information is of help to you. Unfortunately I am currently not able to share any raw data with you.

Having reviewed your protocol,I have a few further questions. How are you performing the standard exactly?Specifically, how are you binding the SAH to the plate?

Ifthe SAH is susceptible to oxidation, I would suggesttrying an alternative method of sample preparation as PCA is an oxidizing agent. It may even be worth adding a reducing agent to your samples such asDDT or ß-mecaptoethanol. AlthoughI would not suggest using these in the antibody diluent buffers as it may reduce the disulphide bonds and reduce the activity.