Overview

  • Product name
    Anti-Insulin degrading enzyme / IDE antibody
    See all Insulin degrading enzyme / IDE primary antibodies
  • Description
    Rabbit polyclonal to Insulin degrading enzyme / IDE
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, IHC-FoFr, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Cow, Cat, Dog
  • Immunogen

    Synthetic peptide derived from within residues 950 to the C-terminus of Human Insulin degrading enzyme/ IDE.

    Read Abcam's proprietary immunogen policy (Peptide available as ab32215.)

  • Positive control
    • Mouse and Rat brain lysate. Mouse and Rat hippocampus lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab32216 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
WB Use a concentration of 1 µg/ml. Detects a band of approximately 118 kDa (predicted molecular weight: 118 kDa).Can be blocked with Human Insulin degrading enzyme / IDE peptide (ab32215).
IHC-P 1/50. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-FoFr 1/100.
IHC-Fr 1/500.

Target

Images

  • Lane 1: Wild-type HAP1 cell lysate (20 µg)
    Lane 2: Insulin degrading enzyme / IDE knockout HAP1 cell lysate (20 µg)
    Lane 3: K562 cell lysate (20 µg)
    Lane 4: HepG2 cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab32216 observed at 120 kDa. Red - loading control, ab8245, observed at 37 kDa.


    ab32216 was shown to specifically react with Insulin degrading enzyme / IDE in wild-type HAP1 cells. No band was observed when Insulin degrading enzyme / IDE knockout samples were examined. Wild-type and Insulin degrading enzyme / IDE knockout samples were subjected to SDS-PAGE. ab32216 and ab8245 (loading control to GAPDH) were diluted at 1μg/ml and 1/10,000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

  • Immunofluorescent staining for Insulin degrading enzyme/IDE in rat brain rat hippocampus using Rabbit polyclonal to Insulin degrading enzyme/IDE (ab32216). . The staining is located in the neuronal soma and is finely punctuated. The picture was acquired using the X20 objective. Protocol details: Rats were intracardially perfused with 4% paraformaldehyde. Whole brain tissue was post-fixed overnight in the same fixative, and cryoprotected in 20% sucrose and frozen in OCT. 30µm coronal sections were cut by cryostat for use in fre floating IHC. Primary antibody ab32216 was incubated overnight at 1/100 at room temperature. Secondary antibody Alexa fluor 488 1/1000 was incubated for 2 hours at room temperature.
  • Image courtesy of Human Protein Atlas

    ab32216 staining human Gall bladder. The paraffin embedded human skin tissue was incubated with ab32216 (1/50 dilution) for 30 mins at room temperature. The staining is predominantly cytoplasmic although some nuclear staining can be seen. Antigen retrieval was performed by heat induction in citrate buffer pH 6. Ab32216 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.

    Further results for

  • All lanes : Anti-Insulin degrading enzyme / IDE antibody (ab32216) at 1 µg/ml

    Lane 1 : Mouse Brain at 20 µg/ml
    Lane 2 : Brain (Rat) Whole Cell Lysate - normal tissue at 20 µg
    Lane 3 : Mouse Hippocampus Lysate at 20 µg
    Lane 4 : Rat Hippocampus Lysate at 20 µg

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 118 kDa
    Observed band size: 118 kDa

References

This product has been referenced in:
  • Fu W  et al. Tongluo Xingnao effervescent tablet reverses memory deficit and reduces plaque load in APPswe/PS1dE9 mice. Exp Ther Med 15:4005-4013 (2018). Read more (PubMed: 29563991) »
  • Khodadadi D  et al. Treadmill Exercise Ameliorates Spatial Learning and Memory Deficits Through Improving the Clearance of Peripheral and Central Amyloid-Beta Levels. Neurochem Res 43:1561-1574 (2018). Read more (PubMed: 29948724) »
See all 35 Publications for this product

Customer reviews and Q&As

1-10 of 14 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Brainstem and kidney)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid
Permeabilization
No
Specification
Brainstem and kidney
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 2% · Temperature: 21°C
Fixative
Formaldehyde

Mr. Carl Hobbs

Verified customer

Submitted Sep 26 2016

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this antibody did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued an aternative free of charge replacement of ab133561 with the order number 1213991.

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 me know.

I wish you the best of luck with your research.

Read More

Answer

Thank you for your reply.

I would like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of ab32216 IDE antibody or this batch. Regrettably, I can suggest you have received a bad vial on this occasion.

Therefore, we would be pleased to provide a replacement from the same antibody, which will still be covered by our guarantee. Alternatively, I fully understand your concerns and if you prefer an alternative replacement of ab133561 or a credit note in this case, I will be pleased to arrange this for you.

I can confirm that ab133561 IDE antibody is tested and guaranteed for both human samples and WB. All tested applications and species covered by the 6 month guarantee are listed on the individual datasheets. The replacement vial will also be covered by the guarantee.

I hope this will be helpful. I look forward to hearing from you with details of how you would like to proceed.

Read More

Answer

Thank you for your message and for providing this further information.

I apologize for the confusion, this is clearer to me now. I am sorry to hear the suggestions made have not improved the results on this occasion. Unfortunately, reviewing the details I am uncertain as to why this antibody is not working in WB for you when it is giving successful results in ICC.

I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement or credit note in compensation.

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

Read More

Question

Order Details
Antibody code: ab32216

Problem
Choose: No signal

Lot number GR1623

Purchase order number #####
or preferably Abcam order number:

General Information
Antibody storage conditions (temperature/reconstitution etc) Aliquoted and stored -20

Description of the problem (high background, wrong band size, more bands, no band etc.) No bands

Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) Human cell extract (SH-SY5Y) stable transformation of choline transporter 1.

Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Lysis buffer: 50mM Tris-HCl pH7.5, 150mM NaCl,1% Triton X-100, Protease inhibitors: 1mM AEBSF, 10ug/ml leupeptin, 25ug/ml aprotinin, 10ug/ml pepstatin A, 700 Units DNAse. Sample buffer (3x): 6% SDS, 0.1875M Tris-HCl pH6.8, 30% Glycerol, 0.015% Bromophenol blue, 7.5% 2-mercaptoethanol. Sample heated 10 mins 55 deg C.

Amount of protein loaded 50 micrograms

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) Reducing (2- mercaptoethanol included in sample buffer), 7.5%

Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) semi-dry transfer (Bio Rad semi-dry transfer cell), PVDF membrane (pre-wet in methanol), Transfer buffer: 14.5g/L Tris-HCl, 7.25g/L Glycine, 20% methanol. Blocking: 8% milk in washing buffer, 1hr. Washing buffer: 1x phoshphate buffered saline pH 7.4, 0.15% Triton X-100.

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) ab32216, rabbit polyclonal to Insulin Degrading Enzyme. Diluted 1/1000 in 8% milk in 1xPBS + 0.15% Triton X-100, overnight, 4 deg C. Washed 2x 30 seconds + 3x 10 minutes in washing buffer.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) goat anti-rabbit-HRP (IgG). 1/10,000 in 1x PBS +0.15% Triton X-100, 1hr room temperature. Washed 2x 30 seconds + 3x 10 minutes in washing buffer.

Detection method (ECL, ECLPlus etc.) ECL

Positive and negative controls used (please specify) Positive controls = successful detection of other antibodies e.g. phospho-PKB (Cell Signalling #4060). Additionally, detection of signal with immunohistochemistry in same cell line.

Optimization attempts (problem solving)
How many times have you tried the Western? Twice. First attempt was after blot had been stripped three times, so repeated with fresh (unstripped) blot.

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?

What steps have you altered?

Additional Notes:

Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

Read More
Answer

Thank you for taking the time to complete our questionnaire.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details:

1. There are inherent difficulties with antibody detection of recombinant tagged proteins that I can recommend considering. This may be a biological artifact rather than a problem with the antibody. Could you confirm that the protein transfected is full length and includes the immunogen sequence? Also, if the recombinant IDE is tagged, it is possible that the tag is preventing access to the antibody. Could you confirm at which region of the protein the tag has been placed?

2. Could you confirm the species from which the recombinant IDE gene was taken?

3. Could you confirm how the stability of transfection has been assessed?

4. I can recommend that due to the points described above, it is important to use an endogenous positive control sample. For example, this antibody has been used successfully on mouse and rat brain lysate.

5. Has the transfer of protein to the membrane and quality of the sample been assessed using a loading control?

6. Was the same antibody used for detection of a signal in the ICC experiments?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details. I hope we can resolve this case for you as soon as possible.

Read More

Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would be pleased to help troubleshoot, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this. It will help you put the information we require together very easily which will enable us to better understand the problem and provide assistance.

I would appreciate if you are also able to provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.

Order Details
Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (brain)
Specification
brain
Fixative
Paraformaldehyde
Antigen retrieval step
Other
Permeabilization
No
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 8% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Feb 28 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (OPC culture)
Specification
OPC culture
Fixative
Paraformaldehyde
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 24°C

Dr. Ruma Raha-Chowdhury

Verified customer

Submitted Feb 10 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (Muscle)
Specification
Muscle
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.3% TritonX in 0.1% PBS
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 24°C

Dr. Ruma Raha-Chowdhury

Verified customer

Submitted Jun 02 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (Brain, liver tissues,)
Loading amount
20 µg
Specification
Brain, liver tissues,
Gel Running Conditions
Reduced Denaturing (12 % Tris Tricine gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Dr. Ruma Raha-Chowdhury

Verified customer

Submitted May 20 2011

1-10 of 14 Abreviews or Q&A

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

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