ChIP Kit (ab500)

Dear Sir or Madam,
I contact you as I am peforming ChIP for the first time, that on tissues.
Working on this specific material, I opted to follow your "Chromatin preparation from tissues for chromatin immunoprecipitation (ChIP)" protocol followed by the "Cross-linking
chromatin immunoprecipitation (X-ChIP) protocol" that I found both on your website.
Possessing an Abcam ChIP kit (ab500), I used the beads of this kit, that I previously blocked a second time.
However, following cautiously the protocols, the results show a high background.
What a bit confusing are differences found in the ab500 kit protocol and in the X-ChIP protocol. Centrifugation amplitudes are quite opposites for some steps and buffers are
quite different too.
Maybe could you help me and enlighten me as to these issues.
Looking forward to hearing from you, I thank you for your time and consideration.

ANSWER:

Thank you for your enquiry.

Sorry to hear you are having trouble with your ChIP experiment.



I have a few additional questions:

1. What specific differences are you referring to between the two ChIP protocols?

2.Did you use a positive control antibody, such as an anti-histone H3 antibody like ab1791?

3. Can you pleasefurther describe the high background or provide data to illustrate the background?

4.Have you made any protocol changes to reduce background levels?



Thanks in advance for the additional information.

As whom it may concern. I am using your ChIP Kit (ab500) and I have just finished some of the components of the kit. I was wondering about the possibility to buy some components separately or if it is possible to know the recipes of the kit's buffers.

ANSWER:

Thank you for contacting us.

At this time the individual kit components for ab500 are not available for separate purchase.

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

Thanks for the reply. I do not add antibody to the negative control, which is why I was hoping the band present at approximately 75kDa was the antibody. Also, seeing as the bands in the upper portion of the western are all consistent across both the sample and negative I was hoping it would be something in the kit that was giving those results. I'm not really sure why it could be the protein I am looking for when no antibody was added? My thoughts were that during the incubation there was degradation that occurred or something. Do you have any other thoughts as to what the upper bands might be seeing as no antibody was added to the negative sample? I probe the western with secondary antibody as well during the western. Would there be this much unspecific binding if the antibody wasn't present?

ANSWER:

Thank you for your reply.

There are only 2 options that I think could explain these results:

1 - The protein is binding to the beads even in the albescence of the antibody, which could be occurring as the beads have been pre-blocked to prevent DNA from binding, not proteins. If this is the case you may have to add a blocking step into your protocol.

2 - The secondary antibody you are using is non-specifically binding. Have you done a secondary only control for your western blot?

Please let me know if there is anything else that I can you with.

Hello,

Thanks for the previous response, I was not able to salvage my protein but I appreciate the help.

I have another question I was hoping you could help me with. As I have mentioned before, I am using the ChIP kit ab500 to isolate the HIF-1alpha protein from some samples I have. My supervisor has asked me to modify this protocol to allow for a western blot to be preformed with the isolated protein rather than following through with the rtPCR as indicated in the protocol. I was in contact with abcam prior to starting this experiment, they told me they couldn't foresee any issues in altering the protocol for use in a western blot but they would not be able to tell me for sure. I decided to go ahead with my proposed protocol and have had many difficulties which I am trying to overcome.

One issue I have is that I need to remove the protein from the protein binding beads in order to run the sample through a gel used in the western blot protocol. To do this, I tried incubating the sample (protein, DNA, protein binding bead complex) in an SDS solution at 98C for 10minutes. This method appeared successful at releasing the samples from the beads but I have not obtained results in my experiments. I was wondering if this temperature is too high for the antibody used and if so, what size the free antibody would appear at on a western? I use antibody ab1 for this protocol. My thought is that this incubation temperature and time are too high and therefore the antibody disassociates from the complex and this is why I cannot detect my protein?

Also, I am questioning whether or not I was able to remove the beads completely from the sample prior to beginning the western blot. If I were to run the beads on the gel, do you know where abouts I would see the corresponding bands and since they too are heated in the incubation, would they degrade and therefore be seen on the gel in the western?

I have attached my experimental results. As you can see there are not many differences between the samples and negative results. I think the bands differing in the samples and negatives are free antibody that disassociated from the complex during the incubation I discussed above. I am having difficulties determining what all the bands are, and am hoping you might be able to help. Please let me know if you have any thoughts on what all the bands are.

ANSWER:

Thank you for your reply.

I will do my best to see if I can help you:

1 - When you analyze your results via PCR rather than western blotting, do you get the correct results (eg no signal in the negative control)? If you have not done this, then I would suggest you try it and make sure the kit is working as it should.

2 - I have attached the document you sent and I have marked what I think are the heavy and light chains of the antibody (50kDa & 25kDa, respectively). Also, I am not completely familiar with this kit, but is there supposed to be antibody in your negative controls?

3 - You do not need to heat your samples to 98C to strip the protein from the beads. Heating the sample to 65/70C for 5 minutes is usually enough.

4 - If you have not removed all of the beads from your sample then they are generally trapped at the top of the gel and do not migrate.

Please let me know if there is anything else I can help you with.

Is recommended sonication time continuous or discontinuous? If discontinuous, does it include cooling periods?

ANSWER:

Thank you for your call earlier this week and for your patience while I have been in touch with the lab about your enquiry.

The results of the U2OS cells shown in the protocol were obtained using a water bath sonicator, and the indicated time corresponds to cycles of 30 seconds on followed by 30 secondsoff at high power. However, the shearing efficiency depends on cell type and the optimal protocol might require additional optimization for each particular cell type. So, if you aren't seeing optimal fragmentation at 15 minutes, you can try longer periods of time.

I hope this information will be useful, but please let me know if you have any additional questions and I'll be happy to help you further.