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RecombinantRabMAb

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

  • Datasheet
  • SDS
Submit a review Q&A (10)References (1)

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Competitive ELISA - Anti-Adenosyl Homocysteine antibody [EPR4499] (ab111903)
  • Anti-Adenosyl Homocysteine antibody [EPR4499] (ab111903)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR4499] to Adenosyl Homocysteine
  • Suitable for: Competitive ELISA
  • Reacts with: Species independent

You may also be interested in

Secondary
Product image
Goat Anti-Rabbit IgG H&L (HRP) (ab205718)

View more associated products

Overview

  • Product name

    Anti-Adenosyl Homocysteine antibody [EPR4499]
    See all Adenosyl Homocysteine primary antibodies
  • Description

    Rabbit monoclonal [EPR4499] to Adenosyl Homocysteine
  • Host species

    Rabbit
  • Specificity

    ab111903 shows the following reactivities with related compounds: S-(5'-Adenosyl)-L-homocysteine: 100% S-(5'-Adenosyl)-L-methionine: 0% Adenosine: 4.4% Homocysteine: 4.4% Cystathionine: 0% L-Cysteine: 1.5% Glutathione: 1.8%
  • Tested applications

    Suitable for: Competitive ELISAmore details
  • Species reactivity

    Reacts with: Species independent
  • Immunogen

    Chemical/ Small Molecule corresponding to Adenosyl Homocysteine conjugated to keyhole limpet haemocyanin.

  • General notes

     

     

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at -20°C. Stable for 12 months at -20°C.
  • Storage buffer

    pH: 7.20
    Preservative: 0.05% Sodium azide
    Constituents: 0.1% BSA, 40% Glycerol (glycerin, glycerine), 9.85% Tris glycine, 50% Tissue culture supernatant
  • Concentration information loading...
  • Purity

    Tissue culture supernatant
  • Clonality

    Monoclonal
  • Clone number

    EPR4499
  • Isotype

    IgG
  • Research areas

    • Signal Transduction
    • Metabolism
    • Amino Acids
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Amino acid metabolism
    • Metabolism
    • Types of disease
    • Cancer

Associated products

  • Alternative Versions

    • Anti-Adenosyl Homocysteine antibody [EPR4499] - BSA and Azide free (ab168537)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab111903 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
Competitive ELISA
1/5000.
Notes
Competitive ELISA
1/5000.

Target

  • Relevance

    Adenosyl Homocysteine is a competitive inhibitor of S-adenosyl-L-methionine-dependant methyl transferase reactions and therefore may play a key role in the control of methylations via regulation of the intracellular concentration of adenosylhomocysteine.
  • Cellular localization

    Cytoplasmic
  • Alternative names

    • S (5' Adenosyl) L homocysteine antibody
    • S Adenosyl L homocysteine antibody
    • S adenosylhomocysteine antibody
    • SAH antibody

Images

  • Competitive ELISA - Anti-Adenosyl Homocysteine antibody [EPR4499] (ab111903)
    Competitive ELISA - Anti-Adenosyl Homocysteine antibody [EPR4499] (ab111903)
    Competitive ELISA: 0.1 µg/ml of BSA-S-adenosylhomocysteine was coated into 96 wells. Serial dilution of S-adenosylhomocysteine (SAH), SAMe, et al. and 0.05 µg/ml of ab111903 were added. HRP conjugated Goat anti-Rabbit IgG antibody was used to develop the color.
  • Anti-Adenosyl Homocysteine antibody [EPR4499] (ab111903)
    Anti-Adenosyl Homocysteine antibody [EPR4499] (ab111903)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (1)

Publishing research using ab111903? Please let us know so that we can cite the reference in this datasheet.

ab111903 has been referenced in 1 publication.

  • Wu C & Tzertzinis G Selection of a mimotope peptide of S-adenosyl-L-homocysteine and its application in immunoassays. Molecules 18:13020-6 (2013). ELISA . PubMed: 24145794

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 10 Abreviews or Q&A

Question

What is the competitive ELISA protocol used with ab111903?

Read More

Abcam community

Verified customer

Asked on Apr 21 2014

Answer

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.

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Apr 21 2014

Question

To Whom It May Concern,
I ordered this antibody for a fluorescent polarization assay without realizing the concentration of the Ab is undetermined. Is there an estimated concentration value available? Even a rough range would help me to getting a starting point of dilution. Thanks.
Sincerely,

Read More

Abcam community

Verified customer

Asked on Feb 20 2013

Answer

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.

Read More

Abcam Scientific Support

Answered on Feb 20 2013

Question

Anti-Adenosyl Homocysteine antibody

Read More

Abcam community

Verified customer

Asked on Mar 20 2012

Answer

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.

Read More

Abcam Scientific Support

Answered on Mar 20 2012

Question

Hi

As well as pursuing the ELISA assay, I'm now looking at alternative methodologies for assaying this molecular and would like to try flow cytometry. So I was wondering what approach Abcam would take to validate and optimise and antibody for flow?
In order to exhaust the ELISA approach my supervisor has asked to enquire as to the possibility of spending a day in your labs with your tech support guys with a view to nailing down the assay and highlighting where the source of problem may be obviously you guys have validated the assay and something between the way you perform the assay and the way we perform the assay is creating a problem. If you can't confirm the possibility of visiting your laboratories then could you please provide me with the contact details of the person in charge of the labs.
Many thanks

Read More

Abcam community

Verified customer

Asked on Mar 20 2012

Answer

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.

Read More

Abcam Scientific Support

Answered on Mar 20 2012

Question

I have two questions about the Anti-Adenosyl Homocysteine antibody (ab111903). What is the concentration of this antibody?  Can I purify this antibody by protein G affinity column? Thanks.

Read More

Abcam community

Verified customer

Asked on Mar 16 2012

Answer

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.

Read More

Abcam Scientific Support

Answered on Mar 16 2012

Question

Hi xxxxx,

Apologises for the delay, I was away on Friday and tied up with interviews yesterday. Please find below the answers to the ELISA questionnaire

xxxxx

1) Abcam product code ab 111903

2) Abcam order reference number or product batch number Lot# GR54919-2

3) Description of the problem No apparent binding of antibody to antigen

4) Type of ELISA: direct, indirect, sandwich competitive ELISA

5) Sample details: NO samples plate coated with SAH-BSA conjugate

6) Target antigen (if found in heterogeneous sample) SAH

7) Positive control

8) Negative control

No primary antibody

No secondary antibody

9) Blocking agent (eg BSA…): Sodium caseinate

Concentration: 2.5%

Blocking time overnight

Blocking temperature 4C

10) Capture or Primary antibody information

Used to coat wells? Yes/no

Species Rabbit monoclonal

Reacts against SAH

Concentration or dilution Range used in checkerboard 1:500 to 1:5000

Diluent buffer PBS based buffer

Incubation time 60min

Incubation temperature 37C

11)Detection antibody:

Species Goat

Reacts against Rabbit

Concentration or dilution Range used in checkerboard 1:1000 to 1:4000

Diluent buffer PBS based buffer

Incubation time 30min

Incubation temperature: 37C

Conjugation HRP

12) Enzyme labeled secondary antibody:

Species

Reacts against

Concentration or dilution

Diluent buffer

Incubation time

Incubation temperature:

Conjugation

13) Washing after primary and secondary antibodies:

Buffer PBS based buffer

Number of washes 4x washes

Length of washes Add & remove no incubation

14) Substrate TMB

Incubation time:3-20mins tried when currently used timings failed to produce blue colour

15) How many times have you run this ELISA? Twice with the listed antibody

Do you obtain the same results every time? Yes

What steps have you altered to try and optimize the use of this antibody? I replaced the SAH-BSA coated plates, the assay itself is an attempt to validate the assay to determine optimal primary and secondary antibody concentrations.

Read More

Abcam community

Verified customer

Asked on Mar 14 2012

Answer

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?

Read More

Abcam Scientific Support

Answered on Mar 14 2012

Question

Hi xxxx

Thank you for the Rabbit anti adenosyl homocysteine antibody(ab111903). I am however experiencing some problems. When using it with my initial secondary antibody or your own Goat anti rabbit HRP antibody I’m not seeing any binding. I’ve checked the system with the initial antibody combination and it is showing binding so I know that there isn’t a problem with the coating on the plates or the TMB substrate. I have tried a checkerboard effect for both primary and secondary antibody dilutions to no avail. Any suggestions?

Read More

Abcam community

Verified customer

Asked on Mar 09 2012

Answer

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.

Read More

Abcam Scientific Support

Answered on Mar 09 2012

Question

Dear xxxx

I am now at the point where I need to think about ordering some more antibody. I am seeing improvements in the system but there is still room for more improvement. I would be interested in trying the alternative antibody that you mentioned. Would it be possible to have a small sample of the antibody in order to test the two antibodies side by side? That way I can make an informed choice regarding which antibody to order.

Many thanks

Read More

Abcam community

Verified customer

Asked on Feb 21 2012

Answer

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.

Read More

Abcam Scientific Support

Answered on Feb 21 2012

Question


I have a question on the Anti-Adenosyl Homocysteine antibody (ab111903) .

The data sheet describe the antibody does not react with SAMe (see attachment). However the figure in the data sheet shows some cross-reaction between the antibody and SAMe. Which one is correct?

Read More

Abcam community

Verified customer

Asked on Feb 15 2012

Answer

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.

Read More

Abcam Scientific Support

Answered on Feb 15 2012

Question

1) Abcam product code ab 35164

2) Abcam order reference number or product batch number:-

Lot number GR28951-2

3) Description of the problem

Lack of sensitivity. Low displacement by antigen within sample or standard

4) Type of ELISA: direct, indirect, sandwich

Competitive antigen (Antigen in sample uses up antibody thus preventing it binding to immobilised antigen bound to plate)

5) Sample details:

Cellular extract (0.4M PCA extracted)

Pure SAH re-constituted in 0.4M PCA

6) Target antigen (if found in heterogeneous sample)

S-Adenosyl homocysteine (SAH)

7) Positive control

Pure SAH in form of standard

8) Negative control

No primary antibody

No secondary antibody

No SAH

9) Blocking agent (eg BSA…): Sodium caseinate: (2 regimes tried)

Concentration: 2.5% in PBS

Blocking time: 37C (4C)

Blocking temperature: 2hr (overnight)

10) Capture or Primary antibody information

Used to coat wells? Yes/no

Species Rabbit

Reacts against SAH

Concentration or dilution Multiple tried in checkerboard, 1:200, 1:300, 1:400 appear best

Diluent buffer Sodium phosphate & Sodium chloride

Incubation time 1hr

Incubation temperature RT & 37C both tried

11)Detection antibody:

Species: Goat

Reacts against: Rabbit

Concentration or dilution 1:2500, 1:1000, 1:500 respectively to primary antibody

Diluent buffer Sodium phosphate & Sodium chloride

Incubation time 30min

Incubation temperature: RT & 37C both tried

Conjugation: HRP conjugated

12) Enzyme labelled secondary antibody:

Species

Reacts against

Concentration or dilution

Diluent buffer

Incubation time

Incubation temperature:

Conjugation

13) Washing after primary and secondary antibodies:

Buffer

Number of washes: 4x250ul

Length of washes: add and remove

14) Substrate: TMB

Incubation time: 3.5min

15) How many times have you run this ELISA? Numerous

Do you obtain the same results every time? Sometimes displacement by antigen in standards is observed and sometimes not. Low levels of displacement with samples

What steps have you altered to try and optimize the use of this antibody?:-

Checkerboard of primary and secondary antibodies

Altering concentration of plate coating with SAH-BSA conjugate

Altering incubation temperatures

Altering incubation times

Read More

Abcam community

Verified customer

Asked on Feb 01 2012

Answer

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.

Read More

Abcam Scientific Support

Answered on Feb 01 2012

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