Overview

  • Product name

  • Description

    Rabbit polyclonal to Dab1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Rat, Chicken, Pig, Zebrafish
  • Immunogen

    Recombinant fragment corresponding to Human Dab1 aa 1-241. Recombinant fragment corresponding to a region within amino acids 1 and 241 of DAB1 (Uniprot ID#O75553)

  • Positive control

    • NT2D1, IMR32, U-87 MG, MCF7 and Mouse brain lysates; HeLa xenograft tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab111684 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 1/1000 - 1/10000. Predicted molecular weight: 60 kDa.
IHC-P 1/100 - 1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Target

Images

  • Anti-Dab1 antibody (ab111684) at 1/10000 dilution + MCF7 whole cell lysate at 30 µg

    Predicted band size: 60 kDa



    7.5% SDS-PAGE.
  • Anti-Dab1 antibody (ab111684) at 1/3000 dilution + Mouse brain cell lysate at 50 µg

    Predicted band size: 60 kDa



    7.5% SDS-PAGE.
  • ab111684 at 1/500 dilution staining Dab1 in paraffin-embedded HeLa xenograft tissue by Immunohistochemistry.

References

This product has been referenced in:

  • Shehabeldin R  et al. Reelin controls the positioning of brainstem serotonergic raphe neurons. PLoS One 13:e0200268 (2018). Read more (PubMed: 30001399) »
  • Sharaf A  et al. Localization of reelin signaling pathway components in murine midbrain and striatum. Cell Tissue Res 359:393-407 (2015). Mouse . Read more (PubMed: 25418135) »
See all 2 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Abreviews
Application
Western blot
Loading amount
60 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (telencephalons)
Specification
telencephalons
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jun 27 2014

Answer

Thank you for getting back to us with that update of your progress. I'm pleased to hear that the serum block has worked well for you.

As you have mentioned, the higher background observed in your blots compared to the ones presented on the datasheet may be due to the different lysates used and their preparation. We would very much appreciate it if you would provide an Abreview for ab111684 detailing the results you have found with the antibody. This will provide future customers with invaluable information on what they can expect and whether the antibody is likely to be suitable for them.

This should only take 5-10 minutes to complete and as a reward you will be awarded with Abpoints which can go towards Amazon vouchers or credit against future Abcam orders. More information on the Abreview system and Abpoints can be found from the following link:

https://www.abcam.com/abreviews

https://www.abcam.com/abpoints

I hope this information has been of help. If you have any further questions, or requests, please do let me know.

Otherwise, I wish you a lovely holiday.

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Answer

Thank you for your recent telephone enquiry, which has been forwarded to me as my colleague Karin is away from the office today. I am sorry to hear you have some concerns regarding the results from ab111684 Anti-Dab1 antibody.

As requested, we have now received the WB testing protocol for this antibody. I hope this will be helpful. However, please note that this woudl be a guideline only and this may require further individual optimization for individual experiments:

The dilution fold used for detection in MCF-7 cells was 1:10000, and in mouse brain is 1:3000.

Blocking condition is 5% non-fat milk at RT for 30-60 mins.

RIPA buffer

Tris-HCl (pH 7.6) 25 mM
NaCl 150 mM
Triton X-100 1 %
Na-deoxycholate 1 %
SDS 1 %


Store at 4 oC. Add the protease inhibitors (see below table) immediately prior use.

Protease inhibitors


Working conc.
PMSF 1 mM
Leupeptin 1 ug/ml
Aprotinin 1ug/ml
Pepstatin 1 ug/ ml

I. Monolayer Cells
1. The following steps should be performed on ice at 4 oC using pre-cold buffer.
Remove culture medium and rinse a subconfluent, 100 mm cell culture plate with PBS twice.
2. Detach Cells with a rubber policeman in 1 ml cold PBS and transfer cell suspension into a 1.5 ml microcentrifuge tube.
3. Pellet cells by centrifuging at 3,000 rpm for 5 min. Remove the supernatant.
4. Suspend the pellet with 1 ml cold RIPA buffer with freshly added protease inhibitors.
5. Allow the tube to stand on ice for 30 min and vortex.
6. Sonication for 5 secs and then stand on ice for 5 secs, Repeat this cycle for 1 min. (Stand on ice in process)
6. Centrifuge the mixture at 20,000 xg at 4 oC for 30 min.
7. Transfer the supernatant to a new tube. The cell lysate can be stored at -80oC.

II. Suspension Cells
1. Collect ˜ 5x107 cells by low-speed centrifugation at RT for 5 min. Carefully remove culture medium.

2. Wash the pellet with PBS at RT, and collect by low-speed centrifugation.

Carefully remove supernatant.

3. Add 1 ml of pre-cold RIPA buffer with freshly added protease inhibitors.
Gently resuspend cells. Incubate at 4 oC for 30 min and vortex.
4. Transfer to microcentrifuge tube(s) and centrifuge at 20,000 xg at 4 oC for 30 min.
5. Transfer the supernatant to a new tube. The cell lysate can be stored at -80oC.
Transfer membrane: Nitrocellulose membrane.

TBST

Tris-HCl (pH 7.6) 20 mM
NaCl 137 mM
Tween 20 0.1 %
Blocking buffer
5% non-fat milk in TBST

III. Antibody incubation

1. Incubate the membrane in blocking buffer at RT for 30˜60 min.
2. Incubate the membrane in blocking buffer with primary antibody at 4 oC overnight.
3. Wash the membrane with TBST for 5 min three times.
4. Incubate the membrane in blocking buffer with secondary antibody at RT for 1 hr.
5. Wash the membrane with TBST for 5 min three times.
6. Follow the instruction of your ECL kit.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee forWB and in mouse 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.

Therefore, if you still continue to have concerns, please do not hesitate to contact us. We will be pleased to help.

Have a good weekend and good luck with your experiments.

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Answer

Thank you for confirming those details.

As requested, I have generated the testing discount code for you as below:

Discount code: xxxxx

Expiration date: 5th November 2012

Please orderab111684 as normalandquote this discountcode in the "Additional comments" section when submitting your resultswith IP in the Abreview for ab111684.Once the Abreview has been submitted the code will become activated and you can quote the code when placing the order for your free product. This code expires on the 5th November 2012. More information on the testing discount scheme can be found form the following link:

https://www.abcam.com/collaboratordiscount

Please do not worry about the email you sent to us. I have marked this as completed. Thank you for flagging this up to me.

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

Until then, I wish you all the best with yourexperiments.

Read More

Question
Answer

Thank you for contacting us yesterday in regards to antibodies to be used in immunoprecipitation of Dab1 from mouse and human samples.

Unfortunately, as discussed over the phone, we do not currently have any antibodies in our catalogue which have been used in immunoprecipitation with both mouse and human samples. The two antibodies we have where they have been shown to work in immunoprecipitation,ab16674 and ab16675, haveonly as yet been tested with mouse samples. The homology of the immunogens used are however very similar and we would therefore expect these antibodies to be able to detect the human protein as well (please refer to the sequence alignment attached).Especially with ab16674 where the homology is 100% (immunogen composed of residues 1-179 of mouse Dab1).

However, as we have not tested these antibodies with human samples we would not be able to guarantee that they would work.If you would like to purchase ab16674or ab16675 and use it with yourhuman samples, we can offer you a testing discount. This offer means that if you purchaseeither of these antibodiesas normal, test the antibody with yourhumansamples and let us know of the results through an Abreview (no matter whether positive or negative) you would be eligible for a free primary antibody of your choice from our catalogue (or the equivalent price of the antibody off any product). More information on this offer can be found here:

www.abcam.com/collaborationdiscount

Please not that the antibody to be tested must be purchased, tested, the Abreview submitted and the free product claimed within a 4 month period. If you would be interested in participating in this scheme please do let me know as a discount code needs to be issued prior to the purchase of ab16674or ab16675.

Alternatively, I would be able to offer the same testing discount if you wanted to try the ab111684 instead. This antibody has been shown to detect both the mouse and human Dab1 protein but has as yet not been used in immunoprecipitation. We would therefore not be able to guarantee that it would work in this application.

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

Read More

Question

Dear Sirs,
I paste here the technical questionnaire about the problems
encountered with Dab1.
I would like also to state that I would wish to exchange
the defective Dab1 antibody with an anti-FoxG1 antibody
(ab18259)
I attach here the results of the test western blot as a
proof.
Since I was asked, I would also like to provide
informations about the Dab1 knockout mouse. Here is an
interesting reference: Howell et al. Neuronal position in
the developing brain is regulated by mouse disabled-1.
1997. Nature. 389:733-7
1) Abcam product code: ab111684

3) Description of the problem:
The band visible after staining is greater than 95 kDa
and it is detectable in both neuronal lysates from
wild-type and Dab1 knockout mice.
4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant
protein…): whole cell lysates from neurons
Lysis buffer : RIPA Lysis Buffer
Protease inhibitors: yes
Phosphatase inhibitors: yes
Reducing agent
Boiling for ≥5 min? yes (5 min)
Protein loaded ug/lane or cells/lane 50 µg per lane
Positive control other established primary antibodies
Negative control lysates from Dab1 knockout neurons
5) Percentage of gel 8%
Type of membrane: Nitrocellulose Membran Hyperbond C Extra
Protein transfer verified yes
Blocking agent and concentration 5% milk PBST
Blocking time: 1h
Blocking temperature: Room Temperature (RT)
6) Primary antibody (If more than one was used, describe in
“additional notes”) :
Concentration or dilution 1:2000
Diluent buffer 5% milk PBST
Incubation time: overnight
Incubation temperature: 4°C
7) Secondary antibody:
Species: donkey
Reacts against: rabbit
Concentration or dilution: 1:5000
Diluent buffer : 5% milk PBST
Incubation time: 1 h
Incubation temperature: RT
Fluorochrome or enzyme conjugate: HRP
8) Washing after primary and secondary antibodies:
Buffer: PBST
Number of washes 3 times
9)Detection method
10) 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.
I hope this was useful.

Read More
Answer

Thank you for confirming these details and for your cooperation. We have received no complaints about ab111684 therefore I am concerned about the results you obtained with it; we currently do not have an explanation for the 95 kDa bands, especially as the other two antibodies showed nice results. The details provided enable us to closely monitor the quality of our antibody and we will investigate this case further.

I am sorry ab111684 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 with FoxG1 antibody ab18259 with the order number 1032809.

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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