Application
ChIP
Sample
Mouse Cell lysate - whole cell (CH12-F3 cell line)
Negative control
CH12-F3 cells AID knock-out
Specification
CH12-F3 cell line
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 8 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde (Fisher #BP531-50
Duration of cross-linking step: 8 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde (Fisher #BP531-50
Positive control
CH12F3 Cells stimulated with cytokine cocktail (also known as CIT) to induce AID expression. Cocktail composition :
+ Recombinant human TGFbeta1 (R&D systems #240-B) 1 ng/mL
+ Recombinant mouse IL-4 (Preprotech #214-14) 10 ng/mL
+ Functional Grade purified a-mouse CD40 (eBiosciences #16-0401) 1 µg/mL
Other product details
Incubation time
16 hour(s) and 0 minute(s) · Temperature: 4°C · Diluent: RIPA Buffer
Additional data
Additional Notes
The Antibody was tested side by side with another commercial Ab against AID that is usually used for ChIP. The positive sample are CH12 cells stimulated with CIT for 16hs where AID should have the highest occupancy on the Igh locus. As negative controls were employed CH12 cells AID KO. Also, an IgG control is included to show the background signal in the amplicons tested by qPCR.
The amplicons tested by qPCR are at the Smu region (P: Promoter, Gb: Gene body) where AID shows the highest occupancy.
The AID occupancy determined with polyclonal Ab ab5197 in positive and negative samples is highly similar, suggesting that the signal detected is indistinguishable from background noise.
Abcam response
Thank you for participating in our AbTrial program and your valuable feedback. As the product had not yet been tested in this species we were unsure what the result might be. Although the homology was very high between human and mouse orthologs, unfortunately the results have not been satisfactory. We appreciate the time and effort that the reviewer has put in to testing this product in this application. We encourage all researchers to contact us should they wish to test any of our products in untested species or applications, as these may qualify for our AbTrial testing program
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Noé Seija
Verified customer
投稿 Jan 15 2021