Anti-Dicer antibody [13D6] - ChIP Grade (ab14601)

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 detect a strong band at ˜ 60kDa for the Dicer protein using the antibody (AB14601), while the molecular weight of this protein is supposed to be 218kDa. Could you please send me the sequence of the
synthetic peptide that has been used to produce this antibody ?
Thanks a lot,.ca

ANSWER:

Thank you for contacting Abcam.
The immunogen sequence for ab14061 is *************.
So that I can better help you with this, would you be able to provide me with a copy of the image that you are getting and also the protocol that you are using, in particular could you include the following information:
1 - What sample type are you using, including species?
2 - How much sample are you loading?
3 - What type of blocking agent are you using?
4 - What primary antibody concentrations have you tried and how long do you incubate for?
5 - How long do you boil your samples for?
I look forward to your reply and helping you resolve this issue.

I have been testing this antibody for two different cell lines:

H293 expressing flag-tagged dice

Embryonic stem cells E14tq2a.

The antibody ordering number: 1040813

Lot nr: GR48699-3

Thank you for your help,

With best regards,

ANSWER:

Thank you for getting back to me promptly and for confirming the sample types used.

As you may be aware, tag itself may obscure native interactions and inhibit the binding of the antibody to the target due to 3-D sterical hindrance.

I could offer you a new vial as a replacement if you wish to test one but it will be from the same batch you have since currently we no not have any new batch/purification in stock.

I would advise you perhaps to use HeLa, MCF7 or human B lymphoma cells as positive control; these should express Dicer at detectable level.

I look forward to hearing from you soon. Please do let me know how you wish to proceed with this enquiry.

Thank you for your email. Here is the protocol I was using for IP:

Immunoprecipitation:

Two lysis buffers were tested:



NP40 lysis buffer





Component



Stock



Final conc.





HEPES-KOH, pH 7.5



1 M



50 mM





KCl



1 M



150 mM





EDTA-NaOH, pH 8.0



1 M



2 mM





NaF



0.5 M



1 mM





NP40 substitute



100 %



0.5 % (v/v)





H2O to 1 liter



n/a



n/a





DTT (fresh)



1 M



0.5 mM





Complete EDTA-free protease inhibitor cocktail (fresh)



n/a



n/a









and RIPA



50mM Tris-cl pH 7.4

150mM NaCl

1% NP40

0.25% Na-deoxycholate

1mM PMSF

1x Roche complete mini protease inhibitor cocktail



1. wash the cells with ice-cold PBS.

2. Drain the PBS, then add ice-cold lysis buffer (1ml per 10cm dish)

3. Scrape the cells with cell scraper and transfer the cell suspension into ependorf tube.

4. Resuspend by pipeting up-down and incubate on ice for 15 minutes.

5.Fragment the DNA by passing the lysed suspension 10 times through a needle attached

to a 1-ml syringe

6. Incubate on ice another 10 min

7. Spin in centrifuge at 14,000 x g at 4°C for 10 minutes.

8. Aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet.

9. Incubate the cell lysate with antibodies (1:50 and 1:100 ware tested) o/n, 4˚C

10. Add dynabeads protein G and incubate for 2h, 4˚C

11. wash 5x with IP buffer and 2x with IP wash buffer.

IP wash buffer







Component



Stock



Final conc.





HEPES-KOH, pH 7.5



1 M



50 mM





KCl



1 M



300 mM





NP40 substitute



100 %



0.05 % (v/v)





H2O to 1 liter



n/a



n/a





DTT (fresh)



1 M



0.5 mM





Complete EDTA-free protease inhibitor cocktail (fresh)



n/a



n/a


I do not think personally it was a technical problem of IP since the control antibodies were working very well, but still I would appreciate any help.

With best regards,

ANSWER:

Thank you for getting back to me promptly and for providing some details.

Would you be so kind to confirm the samples (species, cell type) you are using? It would be much appreciated if you could confirm the batch number, the Abcam order number (or your PON) and the date of purchase so that I can check our system if the delivery was on-time.

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

I have been testing one of your antibodies; anti-Dicer ChIP grade (ab14601, lot nr GR48699-3) for IP, but I wasn’t able to IP any Dicer protein using this antibody. I repeated IP experiment several times, with different condition and different cell lines, but in every case IP result was negative (although my control IP was working perfectly(attachment). Is it possible that something happened to this antibody during the transport or is there a general problem with this lot? I would be grateful for any help. We need urgently a good anti-Dicer antibodies for IP!

With best regards,

ANSWER:

My colleague has passed your enquiry to our Scientific Support Team. I am very sorry to hear that you are having problems with this antibody (ab14601: Anti-Dicer antibody [13D6] - ChIP Grade). Thank you for attaching the IP image to your message and for providing some information.

As the datasheet indicates this product has been specifically tested in ChIP application but not in IP; however both applications are very similar so ab14601 should be working in IP.

Currently, we do not have other antibodies against Dicer which has been characterized specifically for IP.

I could certainly offer you a new vial as a replacement vial if you wish. Before arranging it, I am very happy to take a look at the IP protocol to see if any alterations/optimizations are necessary. Could you provide some further details of the protocol used and complete the following form (attached as a word document).

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.