Anti-Drosha antibody - ChIP Grade (ab12286)

Thank you for sending us a new Drosha antibody. We had a chance to try it out, I have attached our western blot results.
As you will see, we were not able to have a band around 179-190 as per protein molecular weight.
Our fist two lanes are Nuclear extracts and the last two are Cytoplasmic ones. Drosha is only supposed to be in the nucleus, but our cytoplasmic samples are showing plenty of other bands.
What do you think could be happening here? Is there a cleavage that happens and this antibody is showing it around 75KDa? Could we use this band as our reference?
On another related note, we tried the same antibody for immuno-florescence and it worked very beautifully!!! It clearly was staining only on the nucleus.
So this is really strange, as Abcam suggests that this Ab is suitable for both WB and IF, but WB result are really not happening....
Please advice.

ANSWER:

Thank you for your reply.

I am sorry to hear that the replacement antibody didn't work well in WB. I'm not sure why the antibody is showing so many non-specific bands.

We have a different anti-drosha antibody tested in human samples in WB as ab58589. Would you like to give that one a try? Or we can refund your original purchase of ab12286.

http://www.abcam.com/Drosha-antibody-ab58589.html

Please let me know which you would prefer. I look forward to your reply.

human osteosarcoma lysates
same samples, same protocol used before: it worked (other lot)
this lot GR53334-1 not working: no pulldown, very faint WB band

WB: faint band
Ab: 1/1000, 1/200 o/n 4 degree
block: 5% milk 1h
secondary: OK

IP: no pulldown
IP with other Ab to other protein in same complex worked and Drosha was detected
Ab: 3ug/3 mg lysate (worked before), incubation o/n, protein A/G beads used

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 product 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 the order number xxx with ab58589. Please see below for the discount code for testing this antibody in IP.

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 (for WB, human). Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.


DISCOUNT CODE: xxx
Expiration date: xxx

I am very pleased to hear you would like to accept our offer and test ab58589 in IP. This code will give you: 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for IPand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

I wish you the best of luck with your research.

For ab12286, anti-drosha, we're not seeing any bands in human nuclear extracts in WB.

For ab14601, anti-dicer, I'm not seeing a bands at 200 kDa, only 1 band at ˜70 kDa in WB with cytoplasmic extracts.

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 that ab12286 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 of a different lot.

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.


For ab14601, you should try using a gradient gel, or 8% gel. Also try a wet transfer overnight at 4C instead of semi-dry and include SDS to a final concentration of 0.1% and methanol at a max of 10% in the transfer buffer. If you still do not see the band at 200kDa, please let me know.

I wish you the best of luck with your research.

I intend to buy the rabbitpolyclonalantibodies:ab12286to do IHC on human tissues.Could you send me some citiation papers

that usedantibodies:ab12286to do IHC ? Thank you very much.

Look forward to your response.

Sincerely yours,











At2011-11-1022:21:57,technical@abcam.comwrote:
>
>
> DearZongyue,
>
>Wehaveananswertoyourinquiry:
>Thankyouforyourenquiry.
>
>Currently,weonlysellpolyclonalantibodiesagainstDrosha.Isthereanyspecificreasonwhyyouareinterestedinmonoclonalrabbitantibodies?
>
>Wehave2rabbitpolyclonalantibodies:ab12286andab85027andonegoatpolyclonalantibodyab58589.
>
>Ifyouneedanyfurtherassistanceinthefuture,pleasedonothesitatetocontactme.
>
>
>YouroriginalinquirytoAbcam:
>-------------------------------------------
>
> Doyouofferrabbitmonoclonalanti-drosha?
>-------------------------------------------
>
>
>Bestregards,
>Tanya
>
>TanyaBagrij,PhD
>ScientificSupportSupervisor
>Abcamplc
>www.abcam.com
>
>
>AbcamCustomerServicesandTechnicalSupportTeam
>
>www.abcam.com/technical
>[CCE3279082]
>

ANSWER:

Thank you for your enquiry regarding ab12286.
Currently, we have 12 specific publications on this product (ab12286) and the list of these papers can be viewed on the on the on-line datasheet. Since ab12286 has been tested in ICC/IF, IP, WB, ICC, ChIP; most of these articles are WB-related. We do not have any data/publication which would support its suitability for IHC since it has not been characterized yet.
The other two antibodies against Drosha (ab58589 and ab85027) have been tested in IHC-P and we guarantee them in this application but not aware of any IHC-specific published work yet. As soon as we have further information, we will update the datasheets accordingly.
I hope this helps and if I can assist further, please do not hesitate to contact me.

Do you offer rabbit monoclonal anti-drosha ?

ANSWER:

Thank you for your enquiry.

Currently, we only sell polyclonal antibodies against Drosha. Is there any specific reason why you are interested in monoclonal rabbit antibodies? We have 2 rabbit polyclonal antibodies: ab12286 and ab85027 and one goat polyclonal antibody ab58589.

If you need any further assistance in the future, please do not hesitate to contact me.