Anti-c-Myc antibody [9E10] - ChIP Grade (ab32)

AbID: 32, Anti-c-Myc antibody [9E10] - ChIP Grade Rating: Inconclusive Image: Anti-c-Myc antibody [9E10] - ChIP Grade for Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) (Mouse) Sample: Species: Mouse Type: Tissue sections Specification: small and large intestine (APC+/- mouse) Application: Application: Fixative: Formaldehyde Antigen retrieval step: Heat mediated - Buffer/Enzyme Used: Citrate buffer, pH 6 Permeabilization: No Blocking step: 3 % BSA + 2 % serum as blocking agent for 1 hour at 22°C Other detail: Dilution: 1/250 Incubation time: 18 hours at 4°C Diluent: 3 % BSA + 2 % serum in PBS Secondary Antibody: Name: Non-Abcam Antibody was used: Biotinylated anti-mouse IgG (H+L) Conjugation: Biotin Dilution: 1/100 Additional Data: Additional Notes: Staining looks very unspecific no nuclear c-myc in polyps, as it would be expected. ad image: staining of immune cells/plasma cells results from incomplete IgG blocking (as mouse antibody on mouse tissue)

ANSWER:

Thanks for your honest feedback.
It does appear that this vial of ab32 is not working as expected. As per our guarantee, if you have submitted this review within 6 months of receiving this antibody we would be happy to offer a replacement of any primary antibody, credit or refund.

what is the affinity of the antibody?

ANSWER:

Vielen Dank für Ihren Anruf.

In dem folgenden Artikel wurde die Affinität des Antikörpers zu c-Myc tags getestet. Mit ELISA wurde eine Kd von 5.6 "107 für die Sequenz EQKLISEEDLN gemessen.
http://peds.oxfordjournals.org/content/14/10/803.full

Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Attached are the pictures, as discussed on the phone. Thanks for the fast reply and help!
Kind regards,

ANSWER:

Thank you very much for the images and you collaboration!
Please do not hesitate to contact us again, should you have any further question or problems.
I wish you good success with your research!

Phone call: mouse colon and intestin sections, where staining should be, as well as knockout mice with upregulated expression. has only background staining. also when diluting the antibody from 1/100, 1/250 ad 1/500, the staining only gets fainter, but not more specific (will send an image).
Uses mouse on mouse blocking kit and used other antibodies from mouse origin on same tissue blocks with same secondary and had no background - secondary and protocol ok in general.
used no antigen retrieval as well as 2xx10minutes HIER with citrate puffer. Blocking with 2% BSA and 3% horse serum (secondary antibody horse), antibody incubation overnight at 4dC.
No suggestions to make which are expected to significantly improve the results. Minor suggestions would be to use permeabilization, PBST for washings and 1h RT incubation of primary.

ANSWER:

Vielen Dank für Ihren Anruf von gestern.
Es tut mir leid, dass Sie Probleme mit diesem Antikörper hatten. Wie am Telefon besprochen, habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben mit dem alternativen Antikörper ab39688. Sie hat die Referenznummer xx und sollte nächste Woche bei Ihnen ankommen.
Ich wünsche Ihnen viel Erfolg für Ihr Projekt. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

A potential customer would like to detect human Oct4, Sox-2, C-myc in
HFF and HEK293T using these 3 antibodies in western blotting:
AB18976 Abcam Oct4 antibody, 100 ug
AB97959 Abcam SOX2 antibody, 100ug
AB32 Abcam c-Myc antibody [9E10], 100 µg
Please advise if it is feasible?
(I just wanted to double confirm as the customer has used Abcam
filters appropriately)
I look forward to your response!
Many Thanks and Best Regards,

ANSWER:

Thank you for contacting us.

Please advice customer to consult publication whole selecting the antibodies.

HEK293T cells do not express c-Myc and Oct4. They express SOX2.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1289416/

Human embryonic fibroblasts have basal level of Oct4 and Sox2 proteins. You may need to induce the expression of these proteins.

http://sklrm.shsmu.edu.cn/manage/edit/UploadFile/200982102734474.pdf

c-myc is also OK in HEF cells; http://www.pnas.org/content/106/31/12759.full

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