Anti-Dab1抗体(ab111684)

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.

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.

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.

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.

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.