Overview

Description

  • Nature
    Recombinant
  • Source
    Escherichia coli
  • Amino Acid Sequence
    • Accession
    • Species
      Human
    • Sequence
      AVPIQKVQDD TKTLIKTIVT RINDISHTQS VSSKQKVTGL DFIPGLHPIL TLSKMDQTLA VYQQILTSMP SRNVIQISND LENLRDLLHV LAFSKSCHLP WASGLETLDS LGGVLEASGY STEVVALSRL QGSLQDMLWQ LDLSPGC
    • Amino acids
      21 to 167

Specifications

Our Abpromise guarantee covers the use of ab13987 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

  • Applications

    Blocking

    Western blot

    ELISA

  • Purity
    > 95 % SDS-PAGE.

  • Form
    Lyophilised
  • Additional notes
    Store lyophilized protein at -20°C. Aliquot the product after reconstitution to avoid repeated freezing/thawing cycles. Reconstituted protein can be stored at 4°C for a limited period of time; it does not show any change after two weeks at 4°C.
  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

    pH: 7.40
    Constituent: 0.04% Sodium bicarbonate

  • Reconstitution
    Add 0.2 ml of deionized water and let the lyophilized pellet dissolve completely. Once the protein is solubilized, pH can be readjusted to the physiological range. Store lyophilized protein at -20°C

General Info

  • Alternative names
    • FLJ94114
    • LEP
    • LEP_HUMAN
    • LEPD
    • Leptin
    • Leptin (murine obesity homolog)
    • Leptin (obesity homolog, mouse)
    • Leptin Murine Obesity Homolog
    • Leptin Precursor Obesity Factor
    • OB
    • Obese protein
    • Obese, mouse, homolog of
    • Obesity
    • Obesity factor
    • Obesity homolog mouse
    • Obesity Murine Homolog Leptin
    • OBS
    • OTTHUMP00000212285
    see all
  • Function
    May function as part of a signaling pathway that acts to regulate the size of the body fat depot. An increase in the level of LEP may act directly or indirectly on the CNS to inhibit food intake and/or regulate energy expenditure as part of a homeostatic mechanism to maintain constancy of the adipose mass.
  • Involvement in disease
    Defects in LEP may be a cause of obesity (OBESITY) [MIM:601665]. It is a condition characterized by an increase of body weight beyond the limitation of skeletal and physical requirements, as the result of excessive accumulation of body fat.
  • Sequence similarities
    Belongs to the leptin family.
  • Cellular localization
    Secreted.
  • Information by UniProt

References

This product has been referenced in:
  • Mebis L  et al. Contribution of nutritional deficit to the pathogenesis of the nonthyroidal illness syndrome in critical illness: a rabbit model study. Endocrinology 153:973-84 (2012). Blocking . Read more (PubMed: 22166982) »
See 1 Publication for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

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.

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Answer

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!

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Answer

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.

Read More

Question


See below the answer to your question :
Q1: Could you please confirm the batch numbers of ab13987 and ab97051?
ab13987 : GR65976-1
ab97051 : GR44808-2
- Q2: Were these products shipped in the same package?
Yes I think so
- Q3: Could you please provide the Abcam Order Number(s) or your PON?
No we bought them from Sapphirebioscience in Australia, one of my
colleague ordered them, i have not these details.
- Q4: Have you optimized the dilutions of the primary and the
secondary antibody?
I did a dot blot to optimise the secondary dilution because the
recommended dilution of the primary (found in the datasheet) is
1:4000. For this dot blot instead of the 1:4000 dilution, I did a
mistake and use 1:800 dilution. The dot of protein was 300ng, 200 ng,
150ng, 100ng, 75 ng, 50 ng, 25 ng, 12.5ng. For the secondary the
dilution was 1:10000, 1:20000, 1:30000, 1:40000 and 1:50000. After a 2
min exposure, i was able to detect the 12,5 ng of protein with a
secondary dilution of 1:10000 and 1:20000; with the other secondary
dilution, i can detect 25 ng of protein (spot is very light) and 50
ng of protein with the same intensity as the spot obtained with 12,5
ng of protein with a secondary dilution of 1:10000 and 1:20000. Note
that the chemiluminescent detection system used for the dot blot was
more sensitive that the one used for the sensitivity test.
- Q5: Is the HRP still active (ab97051)? Have you used it successfully
with another primary antibody?
I think the antibody is still active because I used it beginning of
march for the dot blot and a week after to do a first western blot
with my protein lysate.
What i have done with this is antibody is:
1- the dot blot as describe above.
2- a western blot with my protein lysate (40ug total protein per lane)
with ab13987 as positive control (300ng), 1:800 dilution for the
primary antibody (I found out my mistake after the incubation),
1:20000 for the secondary (ab97051). I can't detect any band in my
protein lysate but i detected the positive control.
So we decided to do a sensitivity test to know what is the lower
amount of protein that the antibody is able to detect.
3- the sensitivity test
I hope to hear from you soon
Regards

Read More
Answer

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.

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Answer

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.

Read More

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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