Anti-Corticotropin Releasing Factor antibody [4H9] (ab35748)

Hi Abcam,

Please see below an enquiry we have from a customer;

I wanted to inquire whether the CRH antibody ab35748 was directed to only the 41 amino acid mature peptide, not the CRH precursor?

Thank you!

Hope to hear from you soon, and thank you for your help!

Kind regards,

ANSWER:

Thank you for contacting us and your patience while I double checked with the laboratory.

The antibody will recognize an epitope around the Lysine 36 of the mature protein, as the lysine modification blocks the binding completely. The lysine is in position 36 of the corticoliberin - using the full length sequence, it would be the lysine in position 189. http://www.uniprot.org/uniprot/P06850

For information regarding the immunogen used to raise this antibody please see the Kravchenko reference, aslisted on the datasheet, PMID: 7515375 . It is unclear if a peptide or full-length Protein was used as the immunogen; I do not have access to the journal to verify which. As this is a monoclonal antibody, the antibody has only one epitope (the above mentioned).

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

bonjour, voici les conditions utilisées par le service d’anatomopathologie de l’hôpital Cochin voici les différentes techniques utilisées avec l'anticorps CRF :     - démasquage antigénique par la chaleur à pH9 à 99°C pendant 40 min au Bain-marie, anticorps dilué au 1/100 incubation 20 min  à température ambiante(2 cas ont été testés)     - démasquage antigénique par la chaleur à pH9 à 99°C pendant 40 min au Ban-marie, anticorps dilué au 1/50 incubation 2h à température ambiante.  

ANSWER:

Merci pour ces informations.

Je devine qu'un démasquage de l'antigène a également été tenté à pH 6.0 comme recommandé par le laboratoire qui produit cet anticorps, voir mon email du 21 novembre 2011 ci-après. Pourriez-vous s'il vous plait me confirmer ceci?

Le laboratoire vient de m'informer que le tampon recommandé pour le démasquage de l'antigène est un tampon citrate pH 6,0. Placer les lames dans le tampon et chauffer au micro-ondes pendant 10 minutes. Laisser refroidir pendant 15 minutes et rincer avec du tampons TBS.

Préparation du tampon de citrate pH 6.0 (solution 20x): 10,5 g acide citrique dans 250 ml d'eau distillée. Préparation d'une solution 1x : 25 ml de solution 20x 475 ml d'eau distillée Ajuster à pH 6,0 avec NaOH 1M.

antibody is not working, no staining

dilution 1/100

human samples

ANSWER:

Le laboratoire vient de m'informer que le tampon recommandé pour le démasquage de l'antigène est un tampon citrate pH 6,0. Placer les lames dans le tampon et chauffer au micro-ondes pendant 10 minutes. Laisser refroidir pendant 15 minutes et rincer avec du tampons TBS.

Préparation du tampon de citrate pH 6.0 (solution 20x): 10,5 g acide citrique dans 250 ml d'eau distillée. Préparation d'une solution 1x : 25 ml de solution 20x 475 ml d'eau distillée Ajuster à pH 6,0 avec NaOH 1M.

Si vous rencontrez toujours des problèmes avec cet anticorps, nous serions ravis si vous pouviez nous communiquer quelques détails concernant votre protocole en remplissant le questionnaire ci-joint. Ces informations sont très importantes pour nous car elles nous permettent d'investiguer la source du problème rencontré avec cet anticorps et de prendre les dispositions nécessaires pour assurer une bonne qualité de nos produits.

I have ordered the primary antibody "Mouse monoclonal {4H9] to Corticotropin Releasing Factor (ab35748)." I would like to do an immunoabsorption assay and would require the CRF peptide. Can you suggest a few products (CRF peptide) from abcam that my antibody will bind to? Where specifically does this antibody bind to on the CRF peptide?   Thank you for your help.  

ANSWER:

Thank you for contacting Abcam.

Regarding ab35748, this monoclonal antibody was generated using a full length protein as the immunogen.  Unfortunately, epitope mapping has not been performed, although K36 is likely to be a part of the immunogen.  We do not have a specific blocking peptide or recommended protein in our catalog to use for blocking.

I apologize for any inconvenience this may cause.  Please do not hesitate to contact us if you have any additional questions.

Dear Technical support Group!

I have a customer who is not successful with the following antibody and needs some advise:

AB35748,  anti  CRF Corticotropin relaesing factor

Here is her mail:

Hope you are well. I am following up on the ab products bought in June/July this year including ab35748. We have not been able to get this antibody to work at all. I have detailed below the variations that Marilyn in the lab has tried without success. Do you

think you could please get a technical opinion or suggestion from Abcam for us? Or let me know who to contact and I can do that? If there are no technical issues, maybe the ab itself is the problem in which case we would like to find out about replacing it.

Here is a brief report of what I have done did not put in all the steps as I followed the AbCam protocol, what I have reported is the slight variations according to the detection used.

CRF Corticotropin releasing factor [4H9} ab 35748

aliquoted and froze at -20degrees Celcius

First tried on 27-07-2011

Immunohistochemistry as per AbCam protocol

These are my variations

Hydrogen peroxide/methanol 15mins

Blocked with normal goat serum 25mins

Antigen retrieval with 0.01M Citrate buffer in a)pressure cooker  2mins and b)microwave 10mins

Antibody dilutions 1/100, 1/50, 1/20  o/n @ 4 deg. C

Secondary antibody Envision goat anti mouse (Dakocytomation)

Detection with DAB

Tried several times  NO STAINING

Control tissue:Human gastric,small intestine,colon.

Pig tissue: brain,intestine

The tissue was fixed using cold 4% paraformaldehyde. Once fixed for 24 hours it was transferred to 70% ethanol and refridgerated until

it was processed over a 24 hour cycle through 10% buffered formalin, 70% to absolute alcohol, xylol and finally to paraffin wax embedding.

Dont think the antibody is working.

Do you have any suggestions

Sorry cannot get it to work dont know what else to try. Do you want to contact the company and maybe they can offer some other suggestions

Thank you in advance for your help, it is very much appreciated.

Best Regards

ANSWER:

Thank you very much for contacting me again and for passing some useful information.

Although, pig has not been tested for cross-reaction; ab35748 should recognize Corticotropin Releasing Factor in human samples.

I can offer your troubled customer either a new vial as free of charge replacement or a credit note which can be used in future purchase.

Please do let me know how you wish to proceed. I look forward to hearing from you soon.