Anti-Insulin 抗体 [E2E3] (ab9569)

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!

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.

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.

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.

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.

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.  

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.

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.