Anti-Hepcidin-25 antibody (ab75883)

We appreciated the advice we have obteined from Abcam, but I communicate that the use, in our experiments, of 12% gels with MES SDS buffer is equivalent to having done running the proteins at in a gel of approximately 18% polyacrylamide, so we are confident that the protein of our interest has not been lost.

Please let me know if you can send the the immunizing peptide for blocking experiments.

ANSWER:

Thank you for your reply.

I am still concerned that the resolving conditions are not suitable for such a small protein. I will send on the peptide as an exception. However, I am not sure how it will be used for blocking if a band of the appropriate size is not detected.

Please confirm the order number and date of shipping so that I can send on the peptide. Looking forward to your reply.

LOT NUMBER GR57230-1 ORDER NUMBER 3497 DESCRIPTION OF THE PROBLEM more bands and wrong band size: the band we saw (32 kDa) do not correspond to the molecular weight of the protein of interest (2.8 KDa) SAMPLE human cellular lysates and secretomes human tissue extracts human plasma PRIMARY ANTIBODY Abcam / rat and human / BSA 1%-OVA 3% in TBS-Tween 0.1% / dilution 1:1000 / incubation O/N 4°C / washed 3 times for 5 up to 10 min DETECTION METHOD ECL (3 up to 30 minutes of exposition) POSITIVE AND NEGATIVE CONTROLS USED positive controls: HepG2 and MCF-7 lysates human plasma (previously tested by SELDI-TOF mass spectrometry-see attachment) ANTIBODY STORAGE CONDITIONS aliquoted and stored at -20°C SAMPLE PREPARATION Lysis in PLCLB buffer + protease inhibitors. Samples were heated for 10 min at 70°C before gel loading. AMOUNT OF PROTEIN LOADED 30 μg of total proteins ELECTROPHORESIS/GEL CONDITIONS 1) Reducing gel, 4-12% bis-tris, buffer: MOPS SDS 2) Reducing gel 12% bis-tris, buffer: MES SDS TRANSFER AND BLOCKING CONDITIONS 1) TRANSFER conditions: transfer buffer (INVITROGEN), time: 1h30min 2) BLOCKING conditions: BSA 1%-OVA 3% in TBS-Tween 0.1%, time: 1 up to 2 h. We used both PVDF and nitrocellulose membranes SECONDARY ANTIBODY Sigma / rabbit / BSA 1%-OVA 3% in TBS-Tween 0.1% / dilution 1:4000 / incubation 1h RT / washed 3 up to 5 times for 10 min HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 7 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? no steps were altered ADDITIONAL NOTES we would like to test the immunoreactivity of hepcidin synthetic peptide (Hepcidin-25 peptide, ab31875) using the antibody ab75883, which has been used in our experiments. Could you send us an aliquot of this peptide?

ANSWER:

Thank you for contacting us.

I am sorry to hear you have been experiencing problems with this antibody. The qulaity of our products is extremely important to us so thanks for getting in touch.

Hepcidin-25 is a very small protein therefore I would like to suggest some alternative resolving conditions in order to optimize the analysis of small proteins. I think the protein is running off the gel, thus why it is not detected by western blot.

(1) Resolve on an acrylamidegel with 20% acrylamide level
(2) Use Tris-SDS-Page as this is more suitable for small target proteins: http://courses.chem.indiana.edu/C487/documents/NatureprotocolSDSPAGE_000.pdf

Please let me know if this helps improve the results. If this does not help, I would behappy to provide the immunizing peptide for blocking experiments.I look forward to your reply.

Just wanted to let you know that the new antibody worked great! I saw the expected staining in the mouse liver, and I saw some staining in the mouse bladder (which I didn't know what to expect there). Thank you for your excellent service!! I attached a few pics of the liver staining, in case you would like to use them for your website. If so, I can send more details on how I performed the IF staining. Thanks again!

ANSWER:

Thank you very much for your valuable feedback.

We would be very grateful if you could submit an Abreview about this product via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers.

To find out more about our Abreview system, please see the following webpage:

http://www.abcam.com/abreviews

We hope to receive your Abreview shortly. Thank you for your time and effort.

No staining in mouse liver cryosections.

ANSWER:

Thank you for your call today and for letting us know about the trouble with this antibody. As we discussed, I am sending a free of charge vial from a new lot on the order ***, which should arrive tomorrow. Please keep me updated about any results using this new vial, and let me know if there is anything else that we can do for you.