Anti-Peroxiredoxin 1 antibody (ab15571)

no signal with ab15571 lot 1185612
milkor BSA blocking tried
O/N 4ºC
dilution 1/1000
Leucemia cell lines (Human and Mouse)

ANSWER:

Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que le produit ab15571 que vous avez reçu ne fonctionne pas comme attendu.
Le numéro de commande de remplacement gratuitavec leproduit ab41906 est *****. Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

N'hésitez pas à nous contacter lors d'une prochaine occasion.

Please suggest secondary antibodies for ab15571 and ab6276.
Using on rat samples, ICC-IF in double staining.

ANSWER:

Thank you for your telephone enquiry yesterday.

As requested, I am forwarding a list of suitable secondary antibodies for use with ab15571 and ab6276. These have been tested and guaranteed in ICC-IF. I have selected some FITC and PE conjugated antibodies. I can recommend to review the online datasheets for further information.

For the double staining, you will need to select an FITC conjugated secondary for one of the primaries, and a PE conjugated secondary for the other. I have provided a selection of both below, so you can make your own choice.

Secondaries for use with ab6276 mouse IgG1:

FITC conjugated:

ab97239 Goat anti-Mouse IgG1 heavy chain (FITC) secondary antibody
Tested in: Flow Cyt, ICC/IF, IHC-P
http://www.abcam.com/index.html?datasheet=97239 (or use the following: http://www.abcam.com/index.html?datasheet=97239).

ab98692 Goat polyclonal Secondary Antibody to Mouse IgG1 - heavy chain (FITC), pre-adsorbed
Tested in: Flow Cyt, ICC/IF, IHC-P
http://www.abcam.com/index.html?datasheet=98692 (or use the following: http://www.abcam.com/index.html?datasheet=98692).

PE conjugated:

ab99605 Rat monoclonal [SB77e] Secondary Antibody to Mouse IgG1 (PE), pre-adsorbed
Tested in: ELISA, Flow Cyt, ICC/IF
http://www.abcam.com/index.html?datasheet=99605 (or use the following: http://www.abcam.com/index.html?datasheet=99605).


Secondaries for use with ab15571 Rabbit polyclonal:

FITC conjugated:

ab97050 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (FITC)
Tested in: Flow Cyt, ICC/IF, IHC-P
http://www.abcam.com/index.html?datasheet=97050 (or use the following: http://www.abcam.com/index.html?datasheet=97050).

ab97199 Goat polyclonal Secondary Antibody to Rabbit IgG - Fc (FITC)
Tested in: Flow Cyt, ICC/IF, IHC-P
http://www.abcam.com/index.html?datasheet=97199 (or use the following: http://www.abcam.com/index.html?datasheet=97199).

PE conjugated:

ab7007 Donkey F(ab')2 polyclonal Secondary Antibody to Rabbit IgG - H&L (PE), pre-adsorbed
Tested in: Flow Cyt, ICC/IF, IHC-P, IM
http://www.abcam.com/index.html?datasheet=7007 (or use the following: http://www.abcam.com/index.html?datasheet=7007).

ab50677
Goat anti-Rabbit IgG H&L (PE) secondary antibody
Tested in: ICC-IF.
http://www.abcam.com/index.html?datasheet=50677 (or use the following: http://www.abcam.com/index.html?datasheet=50677).

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

We have recently purchased the antibody anti-Peroxirredoxin-1 15571, but unfortunately the antibody is not working properly.

We tested the antibody in different dilutions, 1:10000, 1:5000, 1:1000, in different cells, neurons and lymphocytes and it didn't work.

I would like to know how to solve this problem.



Best Regards,

ANSWER:

We have previously been in contact regarding ab15571, which was not providing satisfactory results.

We provided some troubleshooting tips for the protocol, and I am writing to inquire if these suggestions have helped to improve the results? I would be pleased to receive an update on how these experiments are progressing.

If the antibody is still not working as stated on the datasheet, and the item was purchased within 6 months of contacting us, we would be happy to replace or refund the antibody.

I wish you the best of luck with your research and I look forward to hearing from you. Please do not hesitate to contact us if you have any further questions.

We used reducing conditions. The extracts were made in RIPA. We only use ECF detection in western blot in this center, and it worked properly before.
Unfortunately, we don't have any of these cell lines you referred, so we cannot test it.
Best Regards,

ANSWER:

Thank you for your email.

I am sorry without the positive control it will be hard to determine whether the no band problem is due to antibody, protocol or target being not expressed in cell line you have been using.

We have lysates of these cell lines in catalogue. If you are interested I can send it to you at discounted price.
Human protein Atlas shows this protein is expressed in quite a range of tissue so please click the following link to explore more positive control

http://www.proteinatlas.org/ENSG00000117450/normal

Finally I would like to ask if you have found any publication which shows the protein expression in lymphocytes and HdhQ7/Q7 cells.

I hope this information will be helpful.

Good Morning,
Here is the questionnaire you asked to collect some information about the problems.
Thank you for the attention.
Best Regards,

ANSWER:

Thank you for your enquiry regarding ab15571 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.
We currently have not received any complaint for this product so we are unsure about the cause of recent failure of this product. I appreciate the time you have spent on these experiments in the laboratory and it is disappointing the results have not been successful. The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve your results and that you could consider trying:

- We normally use 5% BSA blocking for 1 hour
- We recommend using this antibody with reducing conditions in WB. The ingredients of sample buffer points towards reducing conditions however you have written non reducing conditions could you specify which info is correct. We recommend reducing and denaturing conditions.
- The antibody gave best resutls wth lysates of HepG2, 293, A549 MCF7, U2OS, and K562 cell line; these lysates can be used an positive control.
- We heat samples in sample buffer at 100C for 10 minutes.
- the dark spot on the membrane could appear after using non filtered dilution and blocking buffer. UI would recommend filtering these.
- Also recomend using RIPA or NP40 lysis buffer with protease inhibitors.
- Have you used the ECF detection system before with other western blot? Could you checkwhether itis compatible.
I am sure following these suggestions will improve results. If however there is no improvement then please do not hesitate to contact us.