Overview

  • Product name
    Anti-Insulin antibody [E2E3]
    See all Insulin primary antibodies
  • Description
    Mouse monoclonal [E2E3] to Insulin
  • Host species
    Mouse
  • Specificity
    The antibody recognizes the biologically most active forms of insulin on the C terminal end. The antibody labels the cytoplasm of beta cells in pancreatic islands and insulinomas (tested on formalin-fixed, paraffin-embedded tissue sections using the Streptavidin-biotinylated peroxidase method).
  • Tested applications
    Suitable for: IHC-P, ELISAmore details
  • Species reactivity
    Reacts with: Cow, Human, Pig, Monkey
  • Immunogen

    Full length protein corresponding to Pig Insulin.

Properties

Applications

Our Abpromise guarantee covers the use of ab9569 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 1/100. Staining of formalin-fixed tissue sections requires treating in boiling 10mM citrate buffer, pH 6.0 for 10-20 minutes. Primary may be incubated for 60 mins at RT.
ELISA Use at an assay dependent concentration. Recommended for insulin monitoring in sera from patients with diabetes or insulin producing tumors.

Target

  • Function
    Insulin decreases blood glucose concentration. It increases cell permeability to monosaccharides, amino acids and fatty acids. It accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in liver.
  • Involvement in disease
    Defects in INS are the cause of familial hyperproinsulinemia (FHPRI) [MIM:176730].
    Defects in INS are a cause of diabetes mellitus insulin-dependent type 2 (IDDM2) [MIM:125852]. IDDM2 is a multifactorial disorder of glucose homeostasis that is characterized by susceptibility to ketoacidosis in the absence of insulin therapy. Clinical fetaures are polydipsia, polyphagia and polyuria which result from hyperglycemia-induced osmotic diuresis and secondary thirst. These derangements result in long-term complications that affect the eyes, kidneys, nerves, and blood vessels.
    Defects in INS are a cause of diabetes mellitus permanent neonatal (PNDM) [MIM:606176]. PNDM is a rare form of diabetes distinct from childhood-onset autoimmune diabetes mellitus type 1. It is characterized by insulin-requiring hyperglycemia that is diagnosed within the first months of life. Permanent neonatal diabetes requires lifelong therapy.
    Defects in INS are a cause of maturity-onset diabetes of the young type 10 (MODY10) [MIM:613370]. MODY10 is a form of diabetes that is characterized by an autosomal dominant mode of inheritance, onset in childhood or early adulthood (usually before 25 years of age), a primary defect in insulin secretion and frequent insulin-independence at the beginning of the disease.
  • Sequence similarities
    Belongs to the insulin family.
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • IDDM antibody
    • IDDM1 antibody
    • IDDM2 antibody
    • ILPR antibody
    • ins antibody
    • INS_HUMAN antibody
    • Insulin A chain antibody
    • Insulin B chain antibody
    • IRDN antibody
    • MODY10 antibody
    • Preproinsulin antibody
    • Proinsulin antibody
    • Proinsulin precursor antibody
    see all

Images

  • Human pancreas stained with ab9569.The antibody labels the cytoplasm of ß cells in pancreatic islands and insulinomas (tested on formalin-fixed, paraffin embedded tissue sections using the Streptavidin-biotinylated peroxidase method).
  • Formalin-fixed, paraffin-embedded monkey pancreas tissue stained for Insulin using ab9569 at 1/800 dilution in immunhistochemical analysis.

    Goat Ani-mouse HRP was used as the secondary antibody.

References

This product has been referenced in:
  • Tian M  et al. Alternative immunomodulatory strategies for xenotransplantation: CD80/CD86-CTLA4 pathway-modified immature dendritic cells promote xenograft survival. PLoS One 8:e69640 (2013). IHC-P . Read more (PubMed: 23922766) »
  • Nykiforuk CL  et al. Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds. Plant Biotechnol J 4:77-85 (2006). WB ; Human . Read more (PubMed: 17177787) »
See all 2 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Monkey Tissue sections (Pancreas)
Antigen retrieval step
None
Permeabilization
No
Specification
Pancreas
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Formaldehyde

Nizar Mourad

Verified customer

Submitted Apr 19 2016

Answer

Thank you very much for your call today and for your enquiry.

As we discussed, please keep me updated about the results with this antibody. Should you have any trouble with it, I will be happy to set up the testing program for you in retrospect.

Please let me know if you have any questions or if there is anything else that we can do for you. Have a nice day!

Read More

Answer

Thank you for your enquiry.
The antibody should be OK. I would recommend using the antibody in an experiment using a positive control sample to make sure it is still active.
Store remainder of antibody at 4 degrees C.
I hope this is helpful. Please contact me again if you have any further questions.

Read More

Answer

Thank you for contacting us. Because the sequence is conserved between the monomeric and hexameric forms of insulin, it would not be possible to get antibodies that would be specific for one form over the other. The easiest way to tell the difference between the two forms would be by their molecular weights in a native WB. For this, I would recommend either ab14042 or ab9569. I hope this helps, please let me know if you need any additional information or assistance.

Read More

Question
Answer

Thank you for your inquiry and I apologize for the delay in this reply.


We do not test this antibody in WB in-house. The only information we have about the use of this antibody is from customer data in the form of an Abreview and reference.


https://www.abcam.com/index.html?datasheet=9569&tab=abreviews&intabreviewid=4206


https://www.abcam.com/Insulin-antibody-E2E3-ab9569-references.html


If you're having difficulty with this antibody in WB, please let us know by providing some protocol information:


1) Abcam order reference number or product batch number

2) Description of the problem

3) Sample preparation:

Type of sample (whole cell lysates, fraction, recombinant protein…):

Lysis buffer

Protease inhibitors:

Phosphatase inhibitors

Reducing agent

Boiling for ≥5 min? yes/no

Protein loaded ug/lane or cells/lane

Positive control

Negative control

4) Percentage of gel

Type of membrane

Protein transfer verified

Blocking agent and concentration

Blocking time

Blocking temperature

5) Primary antibody (If more than one was used, describe in “additional notes”) :

Concentration or dilution

Diluent buffer

Incubation time

Incubation temperature:

6) Secondary antibody:

Species:

Reacts against:

Concentration or dilution

Diluent buffer

Incubation time

Incubation temperature:

Fluorochrome or enzyme conjugate:

7) Washing after primary and secondary antibodies:

Buffer

Number of washes

8)Detection method

9) How many times have you run this staining?

Do you obtain the same results every time?

What steps have you altered to try and optimize the use of this antibody?

Document attachment: Attaching images of your blot is strongly recommended and can greatly speed up our investigation of your problem.


With this information, we will be able to assess any difficulties with the product and offer a replacement or refund if necessary in accordance with our Abpromise. I hope this information helps. Please contact us with any other questions.

Read More

Answer

Thank you for your inquiry. Please excuse the delay of my answer, but I am contacting the source of ab8304 and haven't heard back from them yet. But I am happy to confirm that ab9569 will weakly detect non-reduced insulin (needed to load at least 0.5 ug of purified protein). Also detected reduced or non-reduced proinsulin weakly (need to load at least 0.25 ug of purified protein). I therefore suggest to use reducing condition with this antibody and also recommend to load enough protein. Please excuse the wait, I will get back to you as soon as I receive more information about ab8304.

Read More

Question
Answer

According to our records, ab9569 was proving difficult to use in WB and we were in contact in order to help resolve the issue. Looking at our correspondence, it appears that we are awaiting more details in order to help us better understand the difficulties experienced. If the requested information has already been sent, it appears that it did not reach our Scientific Support team and we apologize for this inconvenience. In this case we would like to ask for the information again so that we can reach a resolution. If the issue has already been settled, please let us know so that we can be assured that the problem has been solved to your satisfaction and update our records. We wish you the best of luck with your research and look forward to a reply.  

Read More

Answer

Thank you for your enquiry. I apologize for the delay in replying to you. It has taken some time to gather all the required information. Sensitivity of antibodies in ELISA assays depends very much on the individual assay procedure and the type of samples. However, in testing I can confirm that the sensitivity of ab9569 Insulin antibody [E2E3] is between 1.0 to 10.0 ng/ml, and the detection level for ab14042 Insulin antibody appears to be ~1 ng/ml. I am sorry we have no further data regarding the minimum detection limits of ab7842 Insulin antibody I hope this information is helpful to you. Should you have any further questions, please do not hesitate to contact us.

Read More

Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal... SAMPLE Porcine Insulin from pancreas (whole molecule) PRIMARY ANTIBODY Capture antibody: ab9569 diluted in phoshate buffer (50mM) diluted to 2 microgram/mL. Incubated first time 2 hours 37C, second time overnight 4C. DETECTION METHOD Conjugated ab used: ab6877. Diluted to manufacturers rules 1:4000. Works well in other tests. POSITIVE AND NEGATIVE CONTROLS USED As a blank I followed the same procedure as mentioned above, but left the antigen incubation behind. A positive control I couldn't perform, because I'm setting this assay up from zero. So the positive control would be this experiment :-} ANTIBODY STORAGE CONDITIONS 4 degrees Celsius TYPE OF ELISA Sandwich ELISA COATING WELL Buffer: Sodium phosphate buffer (50mM) Concentration coating MAb (ab9569): 2 microg/mL BLOCKING CONDITIONS PBS with 1%BSA, for 2 hours at 37 degrees Celsius SECONDARY ANTIBODY Detection antibody: ab14042 diluted in blocking buffer (see above) diluted to 1 microgram/mL. Incubated first time 2 hours 37C, second time overnight 4C (used 2 methods: 1. incubation first with antigen and then added secondary ab, 2. incubation at the same time with antigen). Both methods gave no signal 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? incubation time/temperature of 1st antibody, antigen and 2nd antibody

Read More
Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire and you are largely following an ELISA protocol that we would recommend. There is no reason why these antibodies would not work together in a sandwich ELISA approach. However, we recommend that the capture antibody is seeded at a concentration of 1-10ugml-1. I therefore consider it a useful exercise to increase the concentration of ab9569 to fully determine whether this antibody is not working by ELISA. It may also be useful to seed a plate with the porcine insulin antigen itself and perform a direct approach using an anti-mouse secondary antibody to determine whether it is the antibody or the combinations of antibodies to blame. We have a direct ELISA protocol available at the following link: https://www.abcam.com/assets/pdf/protocols/Direct%20ELISA%20protocol.pdf I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Read More
Application
Western blot
Sample
Arabidopsis thaliana Recombinant protein (total seed extract)
Specification
total seed extract
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Dr. Cory L Nykiforuk

Verified customer

Submitted Feb 24 2006

1-10 of 13 Abreviews or Q&A

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