Anti-Slit2抗体(ab7665)

As discussed the mouse cross reactivity was added due to its citation in PMID 20498081. We unfortunately do not have any other mouse cross reactivity data available.

I have cheked the SLIT2 antibodies from different vendoes. The observed band was 130 kDa with mouse brain lysates.

Please be aware that SLIT2 protein is believed to form 2 cleavage products 130-140 kDa (N product) and 55 kDa (C product). (http://www.uniprot.org/uniprot/Q9R1B9). This antibody may show 130-140 kDa band with mouse lysates. You may need to check the literature about this.

Thank you very much for your email.

The preparation of tissue and cell lysates looks fine to me. The antibody should have worked, I however have following recommendations, I am sure these will help to improve the results.

- Heat lysates in sample buffer 5-10 minutes at 100C.
- ab7665 is a rabbit polyclonal antibody. Please use HRP conjugated ANTI- RABBIT secondary antibody.
- 1% NP-40 lysis buffer will give good results.

Please try these suggestions and let me know if the results do not improve.

Thanks you very much for sending the details and image.

I have reviewed the protocol being used and also have checked the provide image; I am sorry I have few more questions to ask;

- How the cell lysates were prepared? What buffer was used? Was the buffer ice cold? For how long cells left in lysis buffer? have you centrifuged the lysates after preparation?
- How the GUT and Brain lysates were prepared?
- At what temperature the cell lysates were heated in sample buffer and for how long?
- For how long the membrane was blocked?
- Was the membrane (PVDF) activated with methanol/ Transfer buffer?
- What was the secondary antibody?

I have few suggestions for improvement:

- Please avoid touching the membrane with bare hands?
- Prepare the lysates in Ice cold RIPA buffer. The protocol we recommend is; after washing the cells in tissue culture plate with PBS, add lysis buffer (RIPA buffer), scrap of the cells and add into microcentrifuge tube, leave at 4C with constant agitation for 30 minutes, centrifuge at 16,000 g and discard the pellet.
- We recommend heating the lysates in sample buffer for 10 minutes at 100C.
- We recommend blocking the membrane for 1-2 hours in 5%BSA at RT.
- We recommend soaking the membrane for 5-10 minute in transfer buffer.
- We recomend preparing the lysates when cells 70-80% confluent.

Please follow these steps result will be much cleaner. If the result do not improve please do not heasitate to contact em again for further assistance.

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.


I look forward to receiving your reply.

Thank you for contacting us.

The Anti-Slit2 antibody (ab7665) should detect a band at ˜165 kDa indeed, not 55 kDa.

In order to understand the detection of a band at 55 kDa, have you performed a "no primary" control (incubation with the secondary only)? The band at 55 kDa may be due to the secondary antibodyand not the primary.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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I hope this is helpful. Please contact me again if you have any further questions.