Anti-PML Protein antibody (ab53773)

I would like to have more information on your anti PML protein antibody, ref 67761
Host: Rabbit
Tested applications: WB, IHC-P
Cross reactivity: reacts with MOUSE
Immunogen: peptide from N terminal domain of mouse PML protein
There is no image of any western blot on mouse tissue on your website. This is a real key question, as we tested a couple of antibodies, none of them worked correctly on mouse brain tissue, analyzed by western blot. Can you provide me with a western blot from mouse tissue?
Could you give me more information. What is the money back warranty on this antibody?
What is the sequence of the immunogen peptide? to which region (aa number) does it correspond?
looking forward to your answer before placing an order.

ANSWER:

J'ai compris que vous aviez rencontré des difficultés avec deux anti-PML et j'aimerais donc vous rappeler que nos produits sont garantis pour fonctionner tels qu'ils sont décrits sur la fiche technique et que nous pouvons donc toujours résoudre un problèmeen vous remboursant, vous faire un avoir, ou vous envoyer un produit de remplacement.

Pourriez-vous, s'il vous plait, me communiquer les informations suivantes :
- la référence de l'anticorps
- le numéro de lot
- le numéro et la date de la commande
- l'application testée
- le type d'échantillon (espèce, lignée cellulaire, tissu)
- quelques détails sur votre protocole (concentration/dilution de l'anticorps, temps d'incubation, agent bloquant, préparation des échantillons, etc.)

Je serai ravi de vous apporter une solution rapide à votre problème.



Autre point, le laboratoire qui produit l'anticorps anti-PML ab67761 m'a informé que malheureusement aucune image Western Blot n'est actuellement disponible. Si vous souhaitezutiliser ab67761 en Western Blot, il me sera possible de vous offrir une offre promotionnelle. Après achat de l'anticorps et l'envoi d’une Abreview avec vos résultats, vous pourrez commander gratuitement n'importe quel anticorps primaire de notre catalogue.
Etapes à suivre :
1. Nous confirmer que vous souhaiteriezutiliser ab67761en Western Blotafin de recevoir le code de remise correspondant. Important, ce code doit être édité avant l’achat de ab67761.
2.Commander ab67761par téléphone, fax ou internet (http://www.abcam.com). Inutile de mentionner le code sur cette commande.
3. Tester l'anticorps
4. Nous faire part de vos résultats grâce à notre système Abreview. Pour plus d’informations http://www.abcam.com/abreviews
5. Après soumission de votre Abreview, appeler notre service clientèle afin de passer votre prochaine commande grâce au code de réduction. Ce code de réduction est utilisable 120 jours après son édition et utilisable lors de l’achat d’un autre anticorps primaire.

N’hésitez pas à nous demander plus d’informations concernant cette offre promotionnelle.

Termes et Conditions : http://www.abcam.com/collaborationdiscount.

Thank you for your email.
Please arrange a credit note for our customer.

Best regards,

ANSWER:

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.

Credit ID: #####

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

I would like to have more information on your anti PML protein antibody, ref 67761
Host: Rabbit
Tested applications: WB, IHC-P
Cross reactivity: reacts with MOUSE
Immunogen: peptide from N terminal domain of mouse PML protein


There is no image of any western blot on mouse tissue on your website. This is a real key question, as we tested a couple of antibodies, none of them worked correctly on mouse brain tissue, analyzed by western blot. Can you provide me with a western blot from mouse tissue?

Could you give me more information. What is the money back warranty on this antibody?
What is the sequence of the immunogen peptide? to which region (aa number) does it correspond?

looking forward to your answer before placing an order.

Best,

ANSWER:

Merci de nous avoir contactés et désolé pour le délai de ma réponse, j'attendais après le laboratoire les réponses à vos questions.

Concernant l'immunogène : l'information sur la fiche technique est incorrecte. Le peptide utilisé correspond à la régioninterne (entre les acides aminés 400et 500) et non àla région N-terminale. Nous sommes désolés pour cette erreur, la fiche technique va être mise à jour avec la bonneinformation.

J'attends malheureusementtoujours l'image Western Blot pour cet anticorps.

Nous avons2 autresanti-PML garantis pour fonctionner en Western Blot et chez la souris :

ab53773, http://www.abcam.com/ab53773
ab50637, http://www.abcam.com/ab50637



J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

I have attached customer's file.
She tried again following your suggestion but still not satisfied with her results
Please find her data and check the results.
Best regards,

ANSWER:

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange afree of charge replacementor credit note in compensation.

I look forward to hearing from you with details of how you would like to proceed.

LOT NUMBER GR16227-10
DESCRIPTION OF THE PROBLEM Non-specific staining Data shows too much significant signal even in cytoplasm. It has been known that PML is nuclear localized protein. It seems like background effect.
SAMPLE mouse Sample preparation : cells on coverslip
PRIMARY ANTIBODY Concentration or dilution 1:200 Diluent buffer 5% horse serum Incubation time : 4℃ overnight (16hrs) Wash Buffer : 1XPBS Number of washes 3times for 5mins
DETECTION METHOD Confocal710 (ZEISS LSM series)
POSITIVE AND NEGATIVE CONTROLS USED Positive control : non Negative control : non
ANTIBODY STORAGE CONDITIONS -20℃
FIXATION OF SAMPLE Yes/No : Yes If yes: Fixative agent and concentration : PFA 4% Fixation time : 20mins Fixation temperature : Room temperature
ANTIGEN RETRIEVAL Antigen retrieval method : no
PERMEABILIZATION STEP Permeabilization method: with weak detergent Permeabilizing agent and concentration: added 0.5% triton X-100 in PBS
BLOCKING CONDITIONS Blocking agent (eg BSA, serum…): horse serum in PBS Concentration : 5% Blocking time : 1hr Blocking temperature : Room temperature Endogenous peroxidases blocked? no Endogenous biotins blocked? no
SECONDARY ANTIBODY Alex Fluor donkey anti-rabbit-555 (A31572) Invitrogen Species: donkey Isotype: IgG Reacts against: rabbit Concentration or dilution 1:200 Diluent buffer : 5% horse serum Incubation time : 1hr at RT Fluorochrome or enzyme conjugate : alexa 555 Wash Buffer : 1XPBS Number of washes 3times for 5mins
HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3
HAVE YOU RUN A "NO PRIMARY" CONTROL? No
DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes
WHAT STEPS HAVE YOU ALTERED? -
ADDITIONAL NOTES Test for co-localization between PML protein and my target protein in Raw264.7 cells (mouse macrophage) with ICC. I’ve gotten optimized data every time using this protocol what I write above. I don’t think the procedure has some problems. It’s a verified protocol. (Ref. Marnefa et al. 2011. MBoC) NA-related nuclear functions of human Pat1b, the P-body mRNA decay factor) I just wonder why I see some spots even in cytoplasm and I can’t see significant spots called PML body published in previous reports and the review in Abcam webpage. International Immunology, Vol. 23, No. 4, pp. 287–296 doi:10.1093/intimm/dxr004 Advance Access publication 22 March 2011 Molecular Biology of the Celllocalization http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E11-05-0415) on November 16, 2011

ANSWER:

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and also gives us vital information for our monitoring of product quality

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. I would like to reassure you that this antibody is tested and covered by our guarantee forICC-IF and mouse samples.

Before deciding how to proceed with this particular case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some details of the procedure.

1. Could you confirm ifthe current vialofsecondary antibody is working well with other antibodies? It would be important to include a no primary control to assess if this is giving any background.

The speckled non specific fluorescence could be from aggregates of the conjugate. I can recommend to try spinning the vial of secondary verybriefly before using (to spin out aggregates).

The concentration of the secondarymay also need to be reduced in order to help optimize the results.

2. Dust on slides, or particles in the buffers can autofluorescence and can give a speckled staining appearance. I can suggest to ensure the slides and buffers are as clean as possible. Also, this could be caused by cell debris, and so I can recommend to ensure the cell culture sample is as healthy as possible.

3. I can recommend to fix for 10 minutes only to reduce the amount of protein crosslinking, which could affect staining.

4. For the wash steps, try washing in buffer containing 0.2% Tween 20. This will help to wash away any excess antibody. Also include 0.2% Tween 20 in the antibody dilution buffer.

In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested information and details of how you would like to proceed.