His-tagged protein CHIP(chromatin immunoprecipitation) Ni-NTA?

More Juechun Tang's questions See All

Can anyone give suggestion with the binding buffer for immunoprecipitation (or ChIP)??

Hi guys, I need to troubleshoot my IP for low protein in my elutions. I'm performing an IP experiment for a 6X his-tagged protein using an ChIP grade anti-his antibody and dynabeads, I saw...

06 July 2019 9,272 8 View

Did I over-transfer for western blot?

Hi, I used a semi-dry system with 15V 1hr transfer time. The buffer composition was provided by the company who we bought our primary antibody from which is 1.52g Tris, 14.4g Glycine, 100ml...

10 November 2018 1,394 2 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

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Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

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Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

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How to replicate the well conditions of a crystal hit with 20%(w/v) isopropanol?

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Problem with ligating 2.8 kb insert into 8.5 kb plasmid?

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What urea buffer can i use for biotagged protein extraction from streptavidin beads?

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Can I use a protein-devoid condition as my negative control in ELISA?

Hello, I am running a sandwich-type ELISA where I am trying to inhibit a protein-protein interaction using small molecules. For each experiment, I determine my Z' (Z-prime) to determine the...

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Is there a way to extract total RNA and protein from cell culture at the same time, without denaturing protein?

Hi all, Is there a way to extract RNA and protein from the same cell culture sample? Because we need to use the protein for gelatin zymography at later time, we are looking for a method that can...

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Alireza Mordadi

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And take a look at this

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.researchgate.net/post/How_to_remove_bound_DNA_during_protein_purification/amp&ved=2ahUKEwix2PPAtKzeAhUE3KQKHRfEC_AQFjAIegQICBAB&usg=AOvVaw0rA1Y5KsEpsu_I3WLwWj2N&cf=1

Dhiraj Srivastava

I am not an CHIP expert but based on my experience in general protein and protein complex purification, I can say that NiNTA is one of the best and worst resin to use. best because its very cheap and robust. you can even purify denatured protein. however worst because it shows significant non-specific binding to the resin itself. its specifically more prominent with poorly expressed protein. I believe that could be the reason for not using it for any co-IP or pull downs or CHIP kind of experiments because you will get lots of nonspecific hits. by the way, immune-precipitation involves antibody so with any other resin not involving antibody, its better to call it pull-down.

I also found this article by googling chromatin precipitation and NiNTA.

Article Chromatin tandem affinity purification sequencing

also try to search for pull downs and you will find more article. but beware of NiNTA giving non-specific binding.

Juechun Tang

Dhiraj Srivastava Alireza Mordadi Thanks guys, I think non-specific binding might be one of problems. I'm leaning towards getting a good anti-his ab for my CHIP-seq, though Ni-NTA will be much easier.