ChIP Kit - One Step (ab117138)

la concentration de la chromatine peut être mesurée par Bradford ou par un assai BCA.

Notez que nous recommandons d’utiliser entre 5-10 ug de chromatine ce qui correspond à ˜ 0.5 to 1 x 10^6 cellules (la chromatine est généralement composée d’histones et d’ADN dans un ratio d’environ 2:1.)

The wells of ab117138 are coated with protein A/G to bind to the antibody(s) that you use. Multiple antibodies can be used in the same well. However, multiple targeted proteins will be captured, which may make the downstream application and analysis difficult. The best way is to use one antibody for one well in order to gain more specific information regarding which proteins are associated with certain DNA sequences.

The size can be checked by directly running the sheared chromatin as seen in the attached file. If DNA will be purified. The following quick method may be useful: a) Use 10 uL sample and add 40 uL H2O b) Reverse cross-link by adding 2 uL of 5 M NaCl (Final concentration 0.2 M NaCl) c) Boil for 15 minutes d) After returning to room temperature, add 1 uL of 10 mg/ml RNase A at 37 °C for 10 mins e) Clean and purify DNA with PCR Clean-Up Kit, f) Load 1 and 4 uL of sonicated DNA on gel and determine size of smear g) The sonication condition that gives a smear of DNA sizes from 200 bp to 1 kb with a peak around 500 bp (2-3 nucleosomes) should be used for ChIP reactions. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

In general, the chromatin is easy to be sonicated to 200-1000 bps. If the chromatin extracts can not be broken down fully by sonication, it may be due to insufficient time length, power intensity, foaming of chromatin solution during sonication etc. these sonicatio parameters may need to be optimized based on the sonicator type before ChIP. for the best sonication results, we recommend to use water-bath-based sonicators such as Episonic 1100 or covaris, with the established chromatin shearing protocol. DNA purification is not required before ChIP. Cleaned up and eluted DNA after ChIP can be directly used for PCR. However for microarray or sequencing use, the DNA should be further purified as indicated in the user guide.

1. We tested this kit with human samples, but it should also work fine with mouse samples.

2. the primers included in the kit is only for human GAPDH. The following are the suggested primers specific for mouse GAPDH:


F: 5'-TGGAACAGGGAGGAGCAGAGAGCA-3
R: 5'-TACTCGCGGCTTTACGGG-3'

Mouse has been predicted to react, but not yet been tested.

Hello and I apologize for the late reply.

1. The strips are the same for the two
kits.
2. The concentration of chromatin can be measured by
regular protein quantification methods. Histone protein :DNA ratio is
about 2:1.
3. The DNA release buffers are different in these two
kits. the DNA in CP5 of ab11744 requires to be further purified in column and DNA
released from CH3 does not need to be clean up for PCR, which enables ChIP with
the kit ab117138 to be much faster.

Please
let me know if you have any further questions.

L'anti-RNA pol II fournie dans le kit ab117138 est un monoclonal de souris, clone [CTD4H8] qui reconnait la partie C-terminale de toutes les formes de la polymerase (phosphorylée et non-phosphorylée).

J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

Thank you for your reply. I am glad to hear your are able to get the correct band.

Cross linking should not interfere with the IP; however cross-linking is a time-critical procedure. Cross-linking should generally only be carried out for a few minutes. Excessive cross-linking can lead to a decrease in the amount of protein bound to the DNA, reduction in the availability of epitopes/changes in epitopes for antibody binding and, in turn, reductions in the material bound/antigen availability in your sample.It is recommended to always perform a time-course experiment to optimize cross-linking conditions.



There are a number of advantages and disadvantages to both procedures our ChIP tips page details these as well as answers to a number of common ChIP questions. That page is located athttps://www.abcam.com/index.html?pageconfig=resource&rid=310


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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Thank you for contacting us. I am sorry to hear that you have been experiencing issues when in ChIP using this product. According to the Uniprot information for this protein (http://www.uniprot.org/uniprot/Q80TJ7)this product may recognize the ~114 kDa Isoform 1 or the 89 kDa Isoform 2 of PHF8 in mouse. These figures are based on sequence and may not reflect actual size in real world experiments; however this does align with the 111 kDa bands which you have been seeing. While this product has been predicted to work in mouse based on sequence homology, to our knowledge this has not been tested before. I have run a BLAST of the immunogen sequence against mouse with the following result: gi|123285641|emb|CAM17163.1| PHD finger protein 8 [Mus musculus] Length=820 GENE ID: 320595 Phf8 | PHD finger protein 8 [Mus musculus] Score = 46.9 bits (103), Expect = 4e-05, Method: Composition-based stats. Identities = 13/14 (93%), Positives = 13/14 (93%), Gaps = 0/14 (0%) Have you attempted an x-ChIP protocol? We do have a customer review using this antibody in X-ChIP on human cells which you may find under the Abreview tab of the datasheet on the website. X-ChIP may be more suitable when analyzing proteins that have either a weaker DNA affinity or are a long way from DNA. Cross-linking may be required to stop proteins dissociating from the DNA. Histones are tightly associated therefore N-ChIP can be performed when studying histones. It is also possible that there is not enough antibody included in the immunoprecipitation.We would suggest using between 3-5 μg of antibody in the first instance. This could be increased to 10ug if no signal is observed. I have also included our ChIP protocols for your review. We also offer our EpiSeeker One Step ChIP kit which is guaranteed to work in mouse. More information about our EpiSeeker kits and line may be found at these links: https://www.abcam.com/EpiSeeker-ChIP-Kit-One-Step-ab117138.html https://www.abcam.com/index.html?pageconfig=resource&rid=14469 I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. Use our products? Submit an Abreview. Earn rewards! https://www.abcam.com/abreviews

Thank you for contacting us and your interest in our products.

The ChIP kit which your customer is interested in used the protein A beads for isolation and would therefore not be suitable for use with a goat polyclonal antibody. We do however have a different kit that the customer may be interested in. This is the EpiSeeker ChIP Kit - One Step (ab117138). This kit uses a mixture of protein A and G for the immunecapture and would therefore be suitable to use with the goat polyclonal antibody.

I would advise the customer have a look at this kit to see if they think this is suitable for their needs.

I am sorry that I could not be of more help in this instance. If you have any further questions, please do not hesitate to ask