Overview

  • Product name
  • Description
    Rabbit polyclonal to Adiponectin
  • Host species
    Rabbit
  • Specificity
    ab25891 recognises Adiponectin.
  • Tested applications
    Suitable for: IHC-P, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide - a 15 amino acid peptide from near the carboxy-terminus of human adiponectin.

  • Positive control
    • HL60 cell lysate

Applications

Our Abpromise guarantee covers the use of ab25891 in the following tested applications.

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

Application Abreviews Notes
IHC-P Use a concentration of 10 µg/ml.
ICC/IF Use a concentration of 20 µg/ml.
WB Use a concentration of 1 µg/ml. Predicted molecular weight: 26 kDa.Can be blocked with Active human Adiponectin peptide (ab39918).

Target

  • Function
    Important adipokine involved in the control of fat metabolism and insulin sensitivity, with direct anti-diabetic, anti-atherogenic and anti-inflammatory activities. Stimulates AMPK phosphorylation and activation in the liver and the skeletal muscle, enhancing glucose utilization and fatty-acid combustion. Antagonizes TNF-alpha by negatively regulating its expression in various tissues such as liver and macrophages, and also by counteracting its effects. Inhibits endothelial NF-kappa-B signaling through a cAMP-dependent pathway. May play a role in cell growth, angiogenesis and tissue remodeling by binding and sequestering various growth factors with distinct binding affinities, depending on the type of complex, LMW, MMW or HMW.
  • Tissue specificity
    Synthesized exclusively by adipocytes and secreted into plasma.
  • Involvement in disease
    Defects in ADIPOQ are the cause of adiponectin deficiency (ADPND) [MIM:612556]. ADPND results in very low concentrations of plasma adiponectin.
    Genetic variations in ADIPOQ are associated with non-insulin-dependent diabetes mellitus (NIDDM) [MIM:125853]; also known as diabetes mellitus type 2. NIDDM is characterized by an autosomal dominant mode of inheritance, onset during adulthood and insulin resistance.
  • Sequence similarities
    Contains 1 C1q domain.
    Contains 1 collagen-like domain.
  • Domain
    The C1q domain is commonly called the globular domain.
  • Post-translational
    modifications
    Hydroxylated Lys-33 was not identified in PubMed:16497731, probably due to poor representation of the N-terminal peptide in mass fingerprinting.
    HMW complexes are more extensively glycosylated than smaller oligomers. Hydroxylation and glycosylation of the lysine residues within the collagene-like domain of adiponectin seem to be critically involved in regulating the formation and/or secretion of HMW complexes and consequently contribute to the insulin-sensitizing activity of adiponectin in hepatocytes.
    O-glycosylated. Not N-glycosylated. O-linked glycans on hydroxylysines consist of Glc-Gal disaccharides bound to the oxygen atom of post-translationally added hydroxyl groups. Sialylated to varying degrees depending on tissue. Thr-22 appears to be the major site of sialylation. Higher sialylation found in SGBS adipocytes than in HEK fibroblasts. Sialylation is not required neither for heterodimerization nor for secretion. Not sialylated on the glycosylated hydroxylysines. Desialylated forms are rapidly cleared from the circulation.
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • 30 kDa adipocyte complement related protein antibody
    • 30 kDa adipocyte complement-related protein antibody
    • ACDC antibody
    • ACRP30 antibody
    • ADIPO_HUMAN antibody
    • Adipocyte antibody
    • Adipocyte C1q and collagen domain containing protein antibody
    • Adipocyte complement related 30 kDa protein antibody
    • Adipocyte complement related protein of 30 kDa antibody
    • Adipocyte complement-related 30 kDa protein antibody
    • adipocyte-specific secretory protein antibody
    • Adiponectin antibody
    • Adiponectin precursor antibody
    • adiponectin, C1Q and collagen domain containing antibody
    • Adipoq antibody
    • Adipose most abundant gene transcript 1 antibody
    • Adipose most abundant gene transcript 1 protein antibody
    • Adipose specific collagen like factor antibody
    • ADIPQTL1 antibody
    • ADPN antibody
    • APM 1 antibody
    • apM-1 antibody
    • APM1 antibody
    • C1q and collagen domain-containing protein antibody
    • GBP28 antibody
    • Gelatin binding protein antibody
    • Gelatin binding protein 28 antibody
    • Gelatin-binding protein antibody
    see all

Images

  • Lane 1 : Anti-Adiponectin antibody (ab25891) at 0.5 µg/ml
    Lane 2 : Anti-Adiponectin antibody (ab25891) at 1 µg/ml
    Lane 3 : Anti-Adiponectin antibody (ab25891) at 2 µg/ml

    All lanes : HL60 cell lysate

    Predicted band size: 26 kDa
    Observed band size: 26 kDa

  • ab25891 staining adiponectin in rat brain tissue at 10 µg/ml using IHC-P.

References

This product has been referenced in:
  • Matushansky I  et al. A developmental model of sarcomagenesis defines a differentiation-based classification for liposarcomas. Am J Pathol 172:1069-80 (2008). WB ; Human . Read more (PubMed: 18310505) »
  • Rogers PM  et al. Metabolically favorable remodeling of human adipose tissue by human adenovirus type 36. Diabetes 57:2321-31 (2008). WB ; Human . Read more (PubMed: 18599527) »
See all 2 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Question

rift reply. Please see below for more details:

1. Please confirm the order number and date of purchase for this vial? The order number you have kindly provided refers to an order placed in February this year. However, you first contacted us in December. What was the order number for the original vial that did not work? Have you ordered the antibody twice and the results have been unsatisfactory from both vials? Or did you get good results from the first vial before ordering the second vial?
Thank you for your clarification of the circumstances.

Sorry about the confusion. We ordered two lot of the same antibody to try (one ordered in November, one in February). So far we have tried the lot ordered in November (GR59826-2) for many times, but it is still not working nicely as expected. My colleague will try the lot we ordered in February soon.


2. I can suggest it would be beneficial to consider including a no primary control to assess if the secondary antibody is binding non specifically. Has the current vial of secondary antibody been used successfully with other primary antibodies? Have you tried a different secondary antibody?

Although a no primary control was not tested, the secondary antibody we used always give nice bands with other primary antibodies. Please see the attached image 1.


3. Could you confirm the sample has been reduced and denatured in sample buffer containing SDS and mercaptoethanol?

We always use NuPAGE Reducing Agent (containing 500 mM DTT) with 10 min boiling at 100C for reducing conditions. This is working well with other antibodies. Please also see the attached image 1.

4. Has the quality of the sample been assessed using a loading control?

Beta-actin was used as a loading control. Please see image 2 for reference.


Thank you very much indeed.

Best regards,

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Answer

rfor providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacementor credit note in compensation for the vial purchased last November.

Please do not hesitate to let me know how you get on with the vial purchased in February, I hope this works well for you. However, please note we would highly recommend to contact us if there are any problems with a product and let the enquiry come to a resolution before purchasing or being provided withanother vial.

I look forward to hearing from you with details of how you would like to proceed.

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Question
Answer

Thank you for contacting us yesterday in regards to the anti-Adiponectin antibody (ab25891).

As requested, I have attached the IHC protocol used with this antibody. Sepcifically:

Sodium citrate buffer(pH 5.5) was used to perform antigen retrieval (using a microwave to heat the buffer).

5% BSA solution was used to block, incubating the buffer with the tissueovernight at 4°C.

10 µg/mL primary antibody was incubated with the tissue in 1% BSA for 1 hour at room temperature.

I would just like to say however that the conditions may need to be optimised to obtain the best staining for your tissue preparation.

I hope that this information has been of help. If you have any further questions please do not hesitate to contact us again.

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Answer

I'm very glad to hear it. If you do have any problems with any of our antibodies in the future please do let us know as soon as possible and we will always do our best to try and help.

If you would like to participate in our Abreviews system with ab3455, or any of the other antibodies you have purchased from Abcam this will earn you Abpoints which can be used to claim money off future orders or Amazon vouchers. This system allows our customers to review our products, therefore providing vital information to other customers. More information on this scheme can be found from the following link:

https://www.abcam.com/abreviews

Information onhow Abpointscan be used can be found from the following link:

https://www.abcam.com/abpromise

I hope this information has been of help. If you have any further queries or concerns please do let us know.

Until then, I wish you all the best with your research.

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Answer

Thank you for confirming that for me. I have arranged for ab4044 to be sent to you. Unfortunately it is not available in stock at the moment and will therefore take about a week to reach you. I am sorry for this delay and any inconveniencethis causes you. The new order number is xxxxx (purchase order number FOCR xxxxx).

May I ask, on the same order as ab25891 in February you also ordered the anti-adinonectin antibody ab3455. How did you find this antibody to work with?Did youhave any problems using this antibody?

Many thanks for your cooperation.

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Question

Dear xxxxx
Thank you very much for your e-mail from 31.05.12. I filled the complain form as I received the same result as my colleague with another batch of this antibody. I would like to get money back/receive a credit note for this antibody.
Order Details 07.02.2012.
Antibody code: ab25891
Problem Multiple bands
Choose: Non-specific band Multiple bands No signal or weak signal High background
Lot number batch GR10967-5
Purchase order number xxxxxor preferably Abcam order number:
General Information
Antibody storage conditions (temperature/reconstitution etc)
-20C
Description of the problem (high background, wrong band size, more bands, no band etc.)
A lot of bands, not clear if the band of required size is there
Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)
Cell extract
Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)
RIPA buffer, #R0278 Sigma
Inhibitors: NaV, NaF & PMSF 100mM stock, #5726 Sigma
Heating samples at 100C for 5 min
Amount of protein loaded 20 ug/well
Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)
Sample reducing agent 10x stock, #NP009 Invitrogen
mini-protein TGX 4-20% gel, #456-1094 Bio-Rad
Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)
Transfer: Trans-Blot Turbo Blotting system #170-4155, Bio-Rad
with Transfer pack 0.2 PVDF membrane, #170-4156, Bio-Rad - 3 min
The gel was stained with Ponceau stain 0.1% in 1% Acetic Acid - no visible bands
Blocking: 5% Milk (#70166, Sigma) in TBS-T 0.1% for 1 hr at RT
Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
ab25891 Adiponectin Rabbit Ab, 1:1000 in 5% Milk in TBS-T 0.1% at 4C overnight on a rotating wheel
Wash 3x 5 min in TBS-T 0.1%
Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)
#7074 CST goat anti-rabbit, 1:1000 in 5% Milk in TBS-T 0.1% for 2 hrs at RT
Detection method (ECL, ECLPlus etc.)
ECL Plus, #RPN2132, GE Healthcare
Positive and negative controls used (please specify)
Positive - HL-60 cell lysate
Optimization attempts (problem solving)
How many times have you tried the Western? several
Have you run a "No Primary" control? No
Do you obtain the same results every time? Yes
e.g. are the background bands always in the same place? Yes
What steps have you altered?
Additional Notes:
Image: Attached
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asses the results.

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Answer

Thank you for getting back to me with your progress in using the anti-Adiponectin antibody (ab25891).

I am sorry that the use of the new lot has not improved the results sufficiently. Having had a look at the results achieved compared to those initially produced using the antibody purchased in November there has been a marked improvement. Whereas before you had been seeing quite high background from 50 to 100 kDa as well as the expected band at ˜26 kDa, the results now only show minimal background at 50 kDa as well as the expected band (within the LNCap sample). This improvement is probably due to using the milk as the blocking agent instead of the BSA.

It is quite possible that these results can further be improved on by optimising the amount of sample loaded on the gel, the dilution of the secondary antibody and the conditions used to incubate the membrane with the primary antibody. However, I can understand that you have already invested quite a bit of time in optimising this antibody and would thereforebe happy to arrange for a replacement anti-adinonectin antibody to be sent to you(such as https://www.abcam.com/Adiponectin-antibody-19F1-ab22554.html)or any other antibody from our catalogue. Alternatively I can arrange for acredit note to be issued.

Please let me know how you would like to proceed.

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Question
Answer

I now have some more information to share with you in regards to the anti-Adiponectin antibody (ab25891) you are currently using.In performing the western blot presented on the datasheet of this product we used the following protocol:
1. The primary antibody was used at a concentration of 1 ug/mL
2. We used 5% non-fat dry milk in TBS and block overnight at 4°C.
3. Both the primary and secondary antibodies are diluted in the blocking buffer with the primary being incubated with the membrane overnight at 4°C and the secondary for one hour at room temperature.
I hope this information has been of help. Please do let me know if you have been having any difficulty in using the antibody.
Otherwise, I wish you all the best with your research.

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Question
Answer

Thank you for contacting us yesterday.

I have looked into what protocol has been used to perform Western blotting with this antibody previously. I do not have the information of how we have used this antibody in this procedure directly to hand but have sought this information from the lab so I hope I will be able to share this with you soon.

The antibodyhas also been used in two publications:

Matushansky I et al. A developmental model of sarcomagenesis defines a differentiation-based classification for liposarcomas. Am J Pathol 172:1069-80 (2008). WB; Human. PubMed: 18310505

Rogers PM et al. Metabolically favorable remodeling of human adipose tissue by human adenovirus type 36. Diabetes 57:2321-31 (2008). WB; Human. PubMed: 18599527

The former does not make any mention ofwhat conditions were usedin the Western blot but the latter doesstate that a 3%BSA blocking solution was used.

Do you have the results back from the blot which youtried yesterday? How did it look?

I will get back to you as soon as I have further information to share in regards to how the Western blot presented on the datasheet of this antibody. I am sorry for the delay and any inconvenience thisis causing you.

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Answer

Thank you for your reply.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement of alternative antibody ab128870 with the order number 1098022.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

Thank you for your inquiry. I can confirm that HL60 cells express Adiponectin endogenously. We do offer the lysate that was used with ab25891 in our catalogue which could be used as positive control in your case: ab7914: Click here (or use the following: https://www.abcam.com/index.html?datasheet=7914). The cells for the lysate were cultured in RPMI and 10% FBS (Fetal bovine serum). I hope this information is helpful. Please do not hesitate to contact us with any further questions

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