Anti-Hepcidin-25 antibody (ab30760)

LOT NUMBER GR61738-3 ORDER NUMBER xxxxx DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE human cellular lysates and secretomes human tissue extracts PRIMARY ANTIBODY Abcam / rat and human / BSA 1%-OVA 3% in TBS-Tween 0.1% / dilution 1μg/mL / incubation O/N 4°C / washed 3 times for 5 up to 10 min DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED positive controls: HepG2 and MCF-7 lysates 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 times for 10 min 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? none ADDITIONAL NOTES we saw only bands related to the light and heavy chains of the antibody in a WB and no band in other WB. at the time of the delivery, the antibody was not at proper temperature and the package was a bit damaged and lacking of the datasheet

ANSWER:

Thank you for taking time to complete our questionnaire and for contacting us.

I am sorry to hear this antibody is not providing satisfactory results and for the delay in getting back to you. The details provided will enable us to investigate this case and will provide us with vital information for monitoring 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.

In order to understand why this antibody might not be working could I please ask you to answer a few additional questions?

Has the transfer to the membrane been checked with staining of the membrane with a stain such as Ponceau Red?

You say that you are only able to observe light and heavy bands from the antibody, are you performing immunoprecipitation with your sample?

In order to obtain stronger staining I would try incubating the with the primary antibody for 1-2 hours at room temperature. I would also suggest just blocking with 3% milk for 1 hour at room temperature and only add 0.1% milk in your antibody diluents. This may help in obtaining a stronger signal from your sample.

If however these suggestions do not help then please let me know and we can arrange for a replacement antibody or a refund, whichever you would prefer. In regards to how the antibody was delivered to you, we normally deliver at 4°C with an ice pack and this should maintain the activity of the antibody throughout transport. Could you please describe how you received your antibody and I will look into it. Have you ordered your antibody from one of our distributors or directly from us?

I am sorry for the inconvenience this problem is causing.