Recombinant Human Leptin protein (ab13987)

Thank you for passing some information to me.
I understand that ab3583 has been used previously at 1:4000.
Have you had a chance to test this antibody at dilutions of 1/500 or 1/1000 (for 1 hr at room temperature or overnight at 4oC?
Would you prefer to get a new vial of the primary antibody? Please do let me know how you wish to proceed.
I look forward to hearing from you soon.

Thank you for getting back to me and for specifying the sample types.
As you have kindly explained to me in your previous e-mail, the primary and the secondary antibodies worked with the positive control but the signal was very weak using lysates from sheep cardiac tissues.
This antibody (ab3583) should recognize sheep Leptin, the cross-reactivity with this species has been tested and confirmed.
After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:
1) Lysis buffer:
Leptin is secreted so potentially it can be degraded very quickly. It is very important to prepare the samples from fresh tissues and use lysis buffer which contains proteinase inhibitors. The whole process should be carried out on ice. I understand from the initial e-mail that Laemmli buffer was used for lysing the samples. I would rather suggest using RIPA buffer to prevent any unwanted protein degradation in the sheep cardiac tissues. The recipe of RIPA can be found at this website:
https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1
2) Loading amount:
Try to load at least 25-30 ug total tissue lysate per lane onto the gel.
3) Dilution:
You may need to optimize the dilutions of the primary antibody to find out the most suitable working dilution range for sheep cardiac tissue lysate. Brain tissue has high levels of leptin but cardiac tissue may have significantly lower. I have tried to find some information for you regarding the expressed levels of leptin in sheep tissue, but unfortunately not too much data are available on the public sites compared to human, rat our mouse (i.e. Swiss-Prot, Unigen).
http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Oar&CID=524
http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Rn.44444
The expression levels of leptin may vary from cell type to cell type or from tissue to tissue. It would be worth testing 1/500 or 1/1000 (for 1 hr at room temperature or overnight at 4oC).
I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact me again. I would be delighted to help you further.
Have a nice day!

Thank you for confirming the order dfetails.
Would you be so kind to clarify what samples you have been using:
- species,
- cell or tissue types,
- lysate (total cell lysates, or cytoplasmic fraction etc)
I look forward to hearing from you soon.

Thank you for your prompt answers. Your co-operation in this matter is highly appreciated.
I understand that you have already carried out some optimization steps and it seems that the primary and the secondary antibodies worked on positive control but the signal was very weak on the samples. Is that correct? Would you be so kind to clarify what samples you have been using:
- species,
- cell or tissue types,
- lysate (total cell lysates, or cytoplasmic fraction etc)
I have forwarded your response to our Australian Distributor (Sapphire) to get some confirmation about the shipment and order details. Could you please laise with them to find out the Abcam Order number?
Once again, thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Thank you for enquiry and for taking the time to provide some useful details of the experiments. As I understand three of our products (ab3583, ab13987 and ab97051) have been used in these studies.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
- Q1: Could you please confirm the batch numbers of ab13987 and ab97051?
- Q2: Were these products shipped in the same package?
- Q3: Could you please provide the Abcam Order Number(s) or your PON?
- Q4: Have you optimized the dilutions of the primary and the secondary antibody?
- Q5: Is the HRP still active (ab97051)? Have you used it successfully with another primary antibody?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.