Immunoprecipitation - Science method

Hi,

i am having trouble with immunoblotting for HIF1a, and would like to have some tips from you guys. i have tried 4-12% SDS gel, dried/fast and wet transfer both, using Anti-Hif1a from R&D but cannot blot it. These cell do express and have HIF1a proteins. lysis buffer; i am using normal RIPA buffer including PI and phosphotases inhibitors.

would be thankful for your help.

regards  

Hello everyone,

I'm planning a kinase assay and would really appreciate insights, suggestions, or links to protocols/papers that describe similar experiments.

Here’s what I want to do:

My specific questions:

My goal is to test whether the overexpressed kinase can phosphorylate my substrate before proceeding to purify the kinase. The substrate protein is stable and already purified.

If you’ve done something similar or know of published methods, I’d be very grateful for your input!

Thanks in advance for your help.

Best, Kaustubh Prakash

I am doing an immunoprcipitation of a 97KDa protein from mammalian cells. IP is done according to standard protocol (IgG and protein G bead system is used). After IP, when I run a western blot, I see that the protein in the pulldown sample runs at a little but distinctly higher molecular weight than the input sample. It is not a non-specific pull down because the IgG control remains blank. Can someone suggest a possible explanation?

I transfected COS-7 cells with my plasmids of interest and performed co-IP using mouse anti-GFP for IP and mouse anti-myc for IB. I got positive results, confirming that my proteins of interest interact. However, as expected, I also observed two extra bands in the co-IP samples corresponding to the heavy and light chains of the anti-mouse antibody, since both antibodies are from the same species.

To avoid these extra bands, I repeated the experiment using mouse anti-GFP for IP and rabbit anti-myc for IB. However, I observed nonspecific bands in all samples, including co-IP, input, and control (non-transfected), as shown in the image. Does anyone know why this is happening?

I tried blocking with non-fat skim milk and BSA, separately, but the issue persisted.

Thank you in advance!

I am using HEK293 cell line and following this protocol from Abcam:

Up to sonication, my protocol is optimised, and i am getting fragments between 200-500 bp (image attached),

After sonication, I took 100 ul of the sonicated samples and did immunoprecipitation. i used 5ug of Cl8Wg16 antibody against RNA Pol II [it is a test experiment so used to most common possible]

In the end, I used Qiagen PCR purification kit and eluted immunoprecipitated DNA in 30 ul of EB. For samples, i am getting "DNA too low to be detected" and for 2% input control i am getting 5-6 ng/ul of DNA.

please email me Pradip.Karmakar@warwick.ac.uk or reply to this post or message on instagram @pradip_the_great.

thank you in advance,

-Pradip

Hello everyone, does anyone have a protocol for immunoprecipitation and preparation of IP product for mass spectrometry? Specifically using Dynabeads Protein A or G?

Thank you in advance

I am trying to isolate RNA from stress granules, these were obtained using immunoprecipitation, but the amount of RNA and sometimes the quality is not good enough.

Good morning colleagues,

I want to share with you my results, hoping that someone could help me to find a possible explanation.

In the picture you can notice a 1% agarose gel where I run PCR amplicons of a gene promoter. In detail, I started from a best prognosis (left) and a worst prognosis (right) primary cell line, I fixed them with formaldehyde, lysed, extracted the DNA and performed chromatin enzymatic shearing with micrococcal nuclease; then I immunoprecipitated it with histone marks indicated in the lower part of the picture. Ultimately, I designed PCR primers to detect the presence of the amplicon in the immunoprecipitated chromatin.

As you can notice, I can observe the amplicon bands (almost 250bp), but in some histone marks IP I can also see higher bp bands which are difficult to interpretate. I exclude an aspecific amplification, as it doesn't happen in all IP bands and the primers I used should be specific for my region of interest.

Do you have any suggestion or interpretation? Have you ever encountered such a problem in your experience?

Thank you in advance

Jacopo

Lanes 1 to 8 from left to right represent protein from chondrocyte cell line. I ip'd with 5ug of antibody both igg and sox9 from 450ug of total protein. I have no input control as I ran out of protein. I've also attached the coomassie blue staining that I did POST transfer. Thanks again.

1: IP with sox9

2: IP with igg

3: IP with sox9

4: IP with igg:

5: IP with sox9

6: IP with igg

7: IP with sox9

8: IP with igg

Hello all,

I am currently doing Dot Blots to confirm that my antibody is present after an Immunoprecipitant run and for some reason my signal is coming through in the FT and Wash but not in the Elution. My colleague and I are hypothesizing that the antibody we are testing for is stuck to the beads that we are using (AVIDIN for the 1st antibody and HRP for the 2nd) but we are not quite sure if this is the case or if something else is occurring. The kit we are using for Immunoprecipitating is a Thermo Pierce Classic IP kit. We don't know what else to do as we have been tweaking things like the pH for the Elution Buffer and we are kind of at a loss. The next thing we are going to try doing is modifying the wash agent by using 1x PBS but we aren't sure if that will solve anything. Does anyone have insight to this problem or perhaps a similar experience? Sorry this is my first time using ResearchGate so I hope my question comes across clearly.

Hi everyone,

I recently conducted an immunoprecipitation (IP) experiment to isolate my protein of interest, which has a molecular weight of around 25-28 kDa. To minimize interference from IgG chains, I used a TrueBlot secondary antibody. However, I am unsure if my target protein was successfully precipitated, as I still observe strong double bands around the 25-28 kDa range. Additionally, I was unable to detect any co-immunoprecipitated proteins.

Could anyone suggest possible reasons for these issues or provide tips on how to improve my results? Any advice for troubleshooting would be greatly appreciated.

Thank you!

Hello,

I'm looking at immunoprecipitating our flag tagged protein in a chondrocyte cell line derived from mouse. So I need recommendations for a good IP antibody for flag produced in rabbit that could also be used for the subsequent western blot. Any recommendations would be much appreciated. Thank you.

We are currently interested in performing a stimulation protocol on brain slices in order to perform western blotting or immunoprecipitation. However, we do not know how many 300 um slices would be necessary to obtain an adequate protein concentration. If anyone has done something similar or has a reference article I would appreciate the information.

Hello, fellow researchers,

We are currently investigating the role of gut microbiota-derived tryptophan metabolite indole in attenuating inflammation in epithelial cells. Our preliminary findings suggest that indole conjugated to BSA (indole-BSA) interacts with specific cell surface receptors. We aim to identify these receptors by treating cells with indole-BSA, followed by membrane protein isolation and immunoprecipitation.

Challenge and Request for Advice: We are considering the critical step of crosslinking indole-BSA to the membrane receptors before extraction, ensuring that the interactions are preserved during the isolation process. However, we are in need of guidance on the most effective method for crosslinking in this context. Here are our specific questions:

We are open to suggestions, improvements, and any insights that could help refine our approach. We would greatly appreciate your expertise and experiences in similar methodologies or relevant studies.

Thank you in advance for your time and input.

I am planning an immunoprecipitation experiment using Mouse monoclonal [11C9] to Mannan Binding Lectin/MBL (ab26277) antibody to immunoprecipitate for MBL2 in human serum. There are many protocols online for immunoprecipitation utilising cell cultures, where the recommended amount of cells tends to be 10^6 to 10^7, however not so many that utilise human serum.

Reading online, the optimal total protein load of immunoprecipitation seems to be 1-5 mg/mL, with 0.1 mg/mL being the minimum recommended load. Considering that the normal range for total protein in human serum is 60-83g/L (average: 71.5 g/L), loading 5 mg of total protein for immunoprecipitation would mean I need 69.9 mL. Alternatively, loading the minimum (0.1 mg), I would need 1.395 mL of human serum based on my calculations.

I cannot afford to be using 1.395 mL of human serum in my experiment due to lack of sample volume. I was wondering if anyone can share the amount of human serum they've used for immunoprecipitation before, where it was successful. Thank you in advance.

Note: Serum isolated extracellular vesicles using immunoprecipitation (magnetic beads)

I am trying to look at ubiquitination levels for a protein. I use a rabbit polyclonal antibody to immunoprecipitate the protein and then use a mouse monoclonal ubiquitin antibody to do the western. I have tried variations in the protocol but it seems that I always detect bands that have a lower molecular weight than my protein instead of getting higher molecular weight bands that would correspond to the ubiquitinated form of my protein. I tried using a mouse monoclonal antibody for the IP but it gives a lot of non specific bands. I don't have a lot of the polyclonal antibody so I don't know if purifying it would be a good idea. I have tried blocking the beads in 5% BSA, making the antibody-bead complex before adding the lysate but I still get the same results. Does anyone have any suggestions as to how I could modify the IP protocol to get better results?

I conducted a co-immunoprecipitation using protein X antibody to determine if protein X is attached to protein Y. However, the results of my experiments were unusual. To ensure that the procedure was performed correctly, I conducted a control co-immunoprecipitation using protein Y antibody and another antibody that is not specific, which we'll refer to as Z. For the co-immunoprecipitation, I used Dynabeads™ Protein G and confirmed the proteins using WB.

The co-immunoprecipitation experiment using X antibody to pull out Y showed positive results for both X and Y. However, when the experiment was conducted using Y antibody to pull out X, it showed positive results only for Y and no X at all. Additionally, the immunoprecipitation of X and Y using Z antibody (which is nonspecific) showed positive results for both X and Y but the concentration was very weak. This suggests that there might be some issues with the experimental procedure. Can you suggest any measures to confirm the results with proper controls?

Hi there,

I am trying to optimise an immunoprecipitation experiment, where I am attempting to pull down protein X and see if protein Y comes with it. My problem is that I am detecting protein Y in my control IgG IP.

I am using the Pierce magnetic protein A/G beads, lysis buffer [TBS, 150mM NaCl, 1% Triton, protease and phosphatase inhibitors] and wash buffer [TBS, 500mM Nacl, 0.05% Tween]. Lysates are pre-cleaned with blank beads prior to IP. For the experiment I perform a negative control (beads + no antibody), a positive control (beads + rabbit IgG) and an experimental IP (beads + rabbit antibody against protein X). After the IP I perform three washes (1 min each), followed by a water wash, and elution in 60'C 1X laemmli.

When I probe the membrane with a mouse antibody against protein Y, I see a band of the correct molecular weight in the WCE lane and my experimental IP lane, but also in the rabbit IgG lane. The bead-only negative control is clear, so it is not binding to the beads or the tube.

I have validated that the band the protein Y antibody detects by WB (approx. 110 kDa) is specific, using siRNA knockdown. I am limited in that I cannot use another protein Y antibody for detection, so I need to try and fix the issue if possible.

Things I have tried:

Has anyone encountered this problem before? Any suggestions on what to change to remove this non-specific binding to antibodies?

Thanks for any advice you can offer.

Hi. I ran the western blot of my immunoprecipitated sample. I don't know why my protein band appears like a smear?

what could be the possible reason? I loaded 3% of input that is cell lysate and bound protein after IP.

I have attached the blot image. Please suggest me the how to solve this

Hi,

i am getting huge backgrounds from Immunoprecipitation samples when I probe with my antibody of interest from western blot. i am using Dynabeads. Can anyone help me troubleshoot.

thank you

Hello, I have been performing immunoprecipitation assay to understand whether a protein is SUMOylated or not. After overexpression of target protein and extraction of total cell lysate, I carry out immunoprecipitation by targeting the tag of the vector and use SUMO specific antibody for Western blotting. I detect SUMOylation all the while, however, input band and IP band are observed at the same kDa. Also, I check the precipitation on the same membrane by using tag antibody and the detected bands (with both SUMO and tag antibodies) are always overlapped. There is not a shift band even though the protein is SUMOylated. Is there anyone have experienced anything like that before? Is that a normal result for immunoprecipitation detection of SUMOylation? Thanks in advance...

Hi everyone! I'm wondering if anyone here has any experience with the Dynabeads Protein G immunoprecipitation kit (#10007D) from Invitrogen. I'm working on a project that involves immunoprecipitating microsomes from plant tissue using custom-made polyclonal antibodies from the same company. However, I'm having some trouble with the kit. The protein concentration of the purified samples is too low to be measured by the Bradford assay. Most of the proteins seem to stay in the no-binding fraction and don't bind to the magnetic beads. When I do a Western blot with my microsomes, the antibodies can recognize the bands. But when I use them for Dynabeads immunoprecipitation, they don't capture anything. I have tried adding different amounts of Triton X-100 to the microsomes before immunoprecipitation, but it didn't make any difference. Does anyone have any suggestions on how to improve the binding efficiency of the kit? I would really appreciate any advice or tips. Thank you so much!

I want to do MALDI-TOF mass for my protein. For its procedure I need to do bis tris electrophoresis of immunoprecipitated protein, then excise protein band. For running bis tris electrophoresis, I need to solve my protein in LDS sample buffer, but I think that would i replace SDS with LDS in this buffer? Is it too different for doing mass spect?

Can someone explain the difference between a whole cell lysate sample, input sample, and eluate sample when you are running an IP via western blot?

Hello,

Can Alexa Fluor 488 Polyclonal Antibody be used to immunoprecipitate proteins? Has someone tried it or has experience with this?

Many thanks,

Julen.

Hi,

I‘m having an issue with some ChIP experiments I have been doing with a GFP antibody and protein A dynabeads. I’m basically incubating overnight with my antibody and then using protein A dynabeads to pull down the following day. My problem is that when I switch from my binding buffer (50mM HEPES 7.5, 20 mM NaCl, 1mM EDTA, 1% Triton-X-100, 0.1% sodium deoxycholate, protease inhibs) to my first wash buffer (50mM HEPES 7.4, 140 mM NaCl, 1mM EDTA, 1% Triton-X-100, 0.01% sodium deoxycholate) the beads aggregate and clump together but only in my tagged strain and not in my untagged negative control. This persists through all of my washes (0.5M NaCl buffer and 250 mM LiCl buffer) but gets much better when I do the final wash in TE. All of my washes are done at 4 degrees and the buffers were kept on ice. This has repeated several times now.

My qPCR results don’t seem completely off but the background I’m getting at loci where my protein really shouldn’t be (euchromatic regions) is quite high. I think this is because the beads aren’t being washed adequately because of the aggregation.

The pic below shows the aggregation. left is my negative control (untagged IP), right is my tagged strain.

Has anyone else noticed anything like this with dynabeads and found a solution?

any suggestions welcome! Thanks in advance!

Hi,

I am trying to demonstrate a protein-protein interaction through co-IP. To IP, I use a mouse IgG isotype control and a lab-generated mouse antibody to protein X. When I do WB, I use rabbit-generated antibody from Cell Signaling as well as their mouse-anti- rabbit conformation specific secondary (L27A9). In theory, I should not see IgG bands due to using antibodies generated in different hosts and the conformation specific secondary. However, it appears there are IgG bands both in the isotype control and the sample. Shown here, 500ug total protein was used in the IP with 10ug ms antibody. Antigen was allowed to bind beads for 1hr RT while rotating. I eluted in 40 uL total 1x LDS buffer with 5% 2-ME boiled >95C for 10 minutes. My proteins of interest are protein x (~25 kDA with the antibody we use to detect) and protein y (forms various complexes or in free form, ~65 kDA, 55 kDA, 17 kDA free, or 8 kDA free). The input band should be 55 kDA as this is the most prevalent form. My labmates use the same Ms isotype control, ms protein x primary, and secondary with no issues. I have run my protocol by them and they do not see a reason explaining the background.

Lanes L-R: protein x KO cells (IP with protein x), protein x KO cells (IP with IgG), Protein X input (20ug), protein x over-expressing (IP with protein x), protein x over-expressing (IP with IgG), protein x over-expressing input (20ug).

Does anyone have any idea as to why there appear to be heavy and light chain bands here? What can I do to reduce background? Any ideas or advice would be helpful. Thank you in advance!

I am working on immunoprecipitation to check the interaction of proteins, but I found more instance bands in IgG similar to specific Ab after precleaning. Also, I also use different IgG ab and find the same results. Can someone help me to troubleshoot the problem?

Thanks,

Anurag

Dear all,

I am looking for a commercially available pull down assay kit that has conducive buffer conditions and high affinity for the antibody conjugation. My protein of interest (bait) shows a very low binding with the prey and so is there any enhancer that can give good signal for sub-nanomolar concentration ? As I have been using protein A dyna bead from Invitrogen, and every time I see non specific heavy chain of the protein Ig in the western blot. Then to get around the later issue, I used True Blot secondary Antibody, however I can still see huge blob for the Ig heavy chain from the bead. So, if any one can suggest to sort out these two issues with my pull down assay, it would be a great help.

Thank you

With kind regards

Prem

For detection of phosphotyrosine level of a particular protein in a cell lysate, there seem to be two options:

Method two depends on there being available an antibody to the pTyr-modified protein and a dual detection system, such as fluorescence at two wavelengths (since the modified and non-modified bands are likely to overlap on the gel.)

I guess that the immunoprecipitation method is more adaptable to different proteins of interest. Other than that, is one method better than the other (eg greater accuracy)? Is normalisation better in method 2 since you can normalise pTyr signal against protein of interest signal?

I want to overexpress PD-L1 in HEK293 to do immunoprecipitation.

I designed a pcDNA3.1 within N-terminal 6xHis full-length PD-L1 and transfected it into HEK293T by PEI. I used RIPA to get 293 cell lysate. However, I can't get PD-L1 band in WB experiments.

Please help me find some possible solutions.

1. Is TMEM192 the only selectable protein in LysoIP?

2. If I'm going to do LysoIP, should I make sure the TMEM192 protein expression do not change first?

3. After LysoIP, if I want to confirm other protein expression in the lysosomes, which protein can be used as an internal reference when LAMP1 increased after treatment?

Hi, I have been doing some immunoprecipitation (IP) work and no matter what I do I cannot get rid of the IgG heavy chain that I see on the western blot. I am blotting for an antibody called XRCC4 which is at 55 kDa, the IgG heavy chain is around 50 kDa and it completely masks everything up. I have bough Clean-Blot™ IP Detection Kit (HRP) which on paper should have helped. However, it has only partially cleaned it up some of the lanes. Would anyone have any further suggestions I could use.

I am trying to detect an endogenous protein of around 135KDa by Coomassie or Silver Stain for a downstream analysis. My immunoprecipitation works well, and the protein seems highly enriched in Western Blot, but I have so far been unable to detect it by staining the SDS gel directly even after pooling multiple lPs. Any suggestion would be highly appreciated.

Hi everyone,

I was just wondering what's the difference between these two detergents in cell lysis buffer. And I am planning to use the cell lysate to do the GFP-trapped pull-down assay.

Hi,

I want to know whether my protein of interest (effector protein from bacteria) undergoes PTM in plant cells. I am thinking of performing immunoprecipitation and detecting the modification on specific amino acids using mass-spec.

As I don't have any experience using mass-spec, I wonder if anybody can recommend me a good commercial service for PTM identification or any protocol for this kind of experiment.

Thank you

Hi, any idea how to analyse the results from IP?

Hi, I am running through chromatin immunoprecipitation. To reverse the cross-link after immunoprecipitation, the protocol calls for an overnight incubation at 65C. Due to some scheduling conflicts I was wondering if it's ok to store the eluted DNA for a couple of days before reversing the cross-link. Does anyone have any insight into this? Thank you.

Hello,

I have performed a FLAG pull-down using anti-FLAG agarose beads. In loading westerns, I have depleted the loading-buffer-boiled lysate that is above the beads after a brief spin. I still have the beads portion of the loading-buffer-boiled lysate. Can I load this portion into SDS-PAGE to extend my sample for more westerns? Will the agarose beads have any negative effect on running the gel besides taking up volume from true lysate?

Thanks!

Hi All:

I am trying to purify an endogenous protein. But the protein is known to have many other interacting proteins in the cell system I am working on. Is there any way I can decrease protein-protein interactions during IP, remove its interacting proteins, and purify my protein of interest alone? Thanks a lot for your help!

We are trying to immunoprecipitate pectin recognized by the antibody JIM7. pH 2.5 glycine does not elute the pectin from the antibody tat is cross linked to a magnetic bead. We hesitate to use high pH elution buffers because they can deesterify and depolymerize pectin. Any suggestions?

As an alternative to the usual IP approach of tagging a protein in cells (trypanosomes) and passing the lysate through affinity columns, which has been time-consuming and unsuccessful for my protein of interest, can I used a recombinant His-tagged protein (tryp protein expressed in bacteria), which I already have, immobilize this on Ni resin and then add cell lysate? Is it likely that protein interactors may bind to the immobilized recombinant protein under the right buffer conditions?

Hi everyone, I'm trying to isolate a protein using immunoprecipitation of a cell lysate. How can I decide if the amount of isolated protein is enough? I do protein quantification for that and the concentration is pretty low. I'll do a western to check if I was able to isolate the protein but with this low concentration of protein, I need a high volume of sample for western blot. So I'm wondering is there any other way to quantify isolated protein? And also is there a specific amount that I am supposed to have? Waiting for your opinions,

Thanks!!

Hello, all! I'm performing an experiment in which I'm doing a CoIP in the presence or absence of RNase to see if a protein-protein interaction is dependent on RNA. I am trying to identify a protein pair that is known to interact in an RNA-dependent manner, particularly in mouse embryonic stem cells (mESCs). I was thinking of trying to identify some proteins involved in translation initiation or spliceosome assembly/function, but am unfamiliar with the biology of these complexes.

Any and all help is greatly appreciated!

Could anyone help me with the following question?

I was trying to purify my protein of interest by immunoprecipitation (IP) and look at the phosphorylation status of the protein using specific phospho-antibodies in normal and diseased condition. But unfortunately my antibody(polyclonal) is not pulling down my protein of interest. I have used IgG as a control to confirm that there was no immunoprecipitation. I am assuming my antibody is not binding to the protein in the 3-D structure but it works very well when I run western. Is there any other way to look at the phosphorylation status of a protein without doing IP.

Thank you in advance!

To successfully form an immune complex in an immunoprecipitation, is it better to incubate the protein lysate (that has the antigen of interest) with the antibody overnight followed by the addition of the antigen-antibody complex to the bead slurry the next day rather than add the antibody to a bead slurry for a couple of hours followed by the addition of the antibody-bead complex to the protein lysate for incubation overnight? Furthermore is it better to use Protein A magnetic beads in comparison to protein A agarose beads for immunoprecipitation experiments?

I want to know what other possible tests you can do to make sure that the normal serum is removed in immunoprecipitation (the one added in preclearing). One test is with the lysis buffer on the preclearing steps. What other checks can be done? TIA

Hi, I'm having some trouble running a basic immunoprecipitation experiment.

I'm trying to pull-down actin from RAW cells, but all I get is a smear throughout the entire lane with no visible or distinct bands (first image).

I ran a second experiment using the same protocol and materials but with pure actin instead of the cell homogenate and was able to pull down actin (see second image).

I'm using RAW 264.7 cell homogenates (obtained from mechanical lysis using a french press), anti-beta-actin antibodies, and protein g sepharose beads. I've been incubating the various proteins rather than a column, but again the second experiment shows that the protocol works.

Thanks for any help and advice.

Why is it important to have an alternative to the lysate preparation, specifically on having a denaturing and a non-denaturing steps for the samples?

Due to an accidental event in our institute and power cutoff I have stored the Dynabead protein G immunoprecipitation kit in -20C for 16-24 hours.

As the kit recommends the storage should be between 2-8 degrees, will the kit work? any suggestions?

I have attempted using DMSO as a drug-delivery vehicle for a water-insoluble drug but it tends to precipitate out after injection into mice. I am considering trying this with a NMP/PEG-300 formulation instead but cannot find any literature of this ever having been done before. Anyone have any insight into this method?

I am trying to immunoprecipitate LKB1 which is a protein with a molecular weight very close to IgG. I want to cross-link my antibody to the beads to help solve this problem.

Is this the best way to do it (crosslinking the antibody to the beads) or is there a more efficient way?

Lets say, an antibody binds a specific region of a protein and this region is involved in interacting with another protein, therefore this epitope region is buried in the hetero-dimer complex. Does this mean my antibody will not recognize the epitope if I do staining or immunoprecipitation? By the way, the region is a coiled-coil domain, and I know that the antibody binds to that region if I denature the proteins and perform Western blotting.

I'm new to mass spec/proteomics and hoping for some advice! 

We want to do mass-spec on the pulldown from an immunoprecipitation.  We are concerned about IgG/antibody contamination in the sample.

Unfortunately, chemically crosslinking (with DSS) our antibody to the beads decreases the activity of our antibody. Silver staining of our IP product shows that we lose a significant amount of target proteins with antibody crosslinking,although it does eliminate IgG elution.

We are looking for a way to reduce IgG elution, without having to crosslink.  Due to budget constraints, we would also like to avoid alternative (expensive) antibody coupling techniques (such as the surface activated Epoxy m270 dynabeads).

I've found some papers suggesting "soft elution"  (https://www.ncbi.nlm.nih.gov/pubmed/21448433) to reduce IgG contamination. Does anyone have experience with this?

Alternatively, is it possible to simply excise the IgG heavy and light chain bands from the gel, and submit the rest of the lane for mass spec?  

Thanks for any help!

Hi all,

I am working on a mammalian protein that is found in the nucleus and I would like to Immunoprecipitate it (IP) and send it for mass spectrometry analysis in order to determine its interaction-partners.

I used several protocols for extraction, but long story short, the insoluble nuclear fraction is salt resistant and not so much is known about those proteins. Theoretically this fraction contains nuclear architecture proteins, nucleolar proteins, as well as some RNPs and a few chromatin proteins. From what I found, the easiest way to work with these proteins is to solubilize them in a buffer containing high concentration of detergent (8M urea, Laemmli loading buffer, high-SDS protein extraction buffer...). However, these strong denaturing conditions are bad for immunoprecipitation.

Concerning IP: my control is a cell line that is knocked out for this protein. When I try to IP it, everything looks correct on my blot. However, if I perform a Ponceau staining after the transfer step, I can see that I IP a lot of proteins even in my KO. I actually have a smear of proteins in both my KO and non-KO conditions (endogenous and tagged-protein), showing that the IP is absolutely not specific.

But it is even worse than just an aspecific antibody issue. I tried to IP the native protein, the tagged version of it, I used magnetic beads, Protein A/G PLUS-Agarose beads, pre-clearing, different types of buffers (including CHAPS-containing buffer, that is used to partially resolubilize membrane proteins) but I cannot make it work. It is really not about any kind of aspecificity.

My guess is that these proteins are so insoluble that they form clumps together, and they will bind to anything you put in the tube, not in a specific way at all. The problem is that to solubilize them, I would have to use detergents, that would then disrupt protein-protein interactions. People in my lab do not have much knowledge about similar samples, so I am quite lost for a while about that. Anyone has worked with similar proteins or know some tips that could help me? I am not even sure that it is something that can be achieved.

Thank you in advance for your response!

Hi,

I have purified my protein of interest using the specific antibody and pulled down the protein-Ab complex with protein A beads. I need to perform mass spec on my protein and need a protocol that can help in the elution. I read about the glycine and urea-based elution method and was wondering if anyone has used these before and has a standardized protocol.

Thank you.

I use the ultracentrifugation protocol to isolate exosomes from cell in culture (BV-2 microglia cell line). I was wondering if there is a viable protocol to isolate specifically microglia-derived exosomes from bloodstream (either through immune-precipitation or even FACS??)

Which marks are the best in this case? (considering that macrophages must be excluded from isolation).

Hello everybody,

In my work, I'm actually interesting about mRNAs-contained in a protein complex in yeast S.cerevisiae.

We perform in the lab the immunoprecipitation of these mRNAs (RIP) from a S.cerevisiae strain for which one of protein complex is tag, and we now wanted to identify these mRNA by sequencing. The mRNA amount control is mRNA immunoprecipitate from an untagged strain.

But how can I determine the number of reads that I have to choose for the mRNA sequencing ?

Thanks !

Best regards

Hi, I am attempting to run an immunoprecipitation and Western blot and just eluted the protein of interest off the beads in 0.1M Glycine at pH 2.67. I reserved the eluate and added Laemmli. It immediately turned yellow, so I add a drop of NaOH and it turned back to blue, however, after boiling, the samples turned colorless and additional NaOH has not turned them back blue. Are these samples ok to run on a gel? TIA!

I’m attempting to co-IP binding partners from a protein. The problem is that no matter what I do, I have ”smudgy” lanes with super high background. This is a problem because I can’t see if my bands of interest appear. I am using near-infrared detection using a Li-cor odyssey scanner and the background appears in both channels, no matter which specific secondary I use.

Protocol info:

My IP protocol is pretty standard from what I can tell. I use pre-cleared GammaBind sepharose beads. Anti-myc antibody to pull down my protein of interest. Elute with a SDS sample buffer that contains bromophenol blue, DTT, B-ME, and various inhibitors. Boil at 95C for 7 minutes, spin down beads, and load the supernatant for SDS-PAGE. I transfer to a nitrocellulose membrane and do pretty standard Western procedures after that.

Troubleshooting so far:

- different beads (protein A/G Plus from Santa Cruz)

- different sample buffer containing different dye (Li-cod’s own with Orange G)

- loading beads that don’t have antibodies doesn’t help

- it’s not a heavy/light chain or antibody compatibility problem

However, NOT boiling the beads at all after adding sample buffer seems to reduce the effect a lot. Still, I really want to know what the problem stems from. We know of other labs that use near infrared detection with the same equipment and very similar protocols and have great success, and they boil their samples. Any suggestions or ideas would be greatly appreciated!

Hello I have prepared acidic glycine and basic trisbase for an immunoprecipitation experiment and was wondering if the solutions can be reused in future immunoprecipitation experiments I want to perform?

We are performing immunoprecipitation (IP) using protein G and protein A/G magnetic beads. We are using tissue protein lysates from chicken intestinal samples. We are trying to separate and identify proteins previously measured through Western Blot analysis. However, after performing SDS-PAGE and Coomasie stain on the eluants of the IP, we are getting a lot of non-specific binding and two prominent bands at ~43 kDa (actin) and ~223 kDa (myosin). One of our controls, IP procedure without antibody at any step, provided the same results as our IgG antibody treatments. Indicating that the proteins eluded from the beads are attaching to the protein G & A/G of the beads.

Why is our IP procedure not precipitating our target antigens and there is non-specific binding to the beads?

For one of my projects, I need to enrich for the lysine-acetylated fraction of a bacterial protein mix.

I'm currently looking for tips and tricks to boost the sensitivity of my IP assay.

The types of samples I use are E. coli cultures, lysed with French-pressing.

As a positive control, I use chemically (fully) acetylated bacterial protein mix.

IP antibodies include a range of antibodies recognizing various acetylated proteins and peptides in WB and ELISA: commercially available rabbit polyclonal anti-AcK, mix of rabbit monoclonal anti-AcK and in-house human IgG monoclonals.

Comparing the WB signal with control proteins, I roughly estimate the abundance of acetylated proteins to be around 0.1-0.3% (50-150 ng/50 ug) of the whole protein mix.

The problem is that all antibodies work well with the positive control (fully acetylated proteins), but they fail to isolate proteins with limited acetylation abundance.

The current protocol I use:

Hi,

I am trying to quantify proteins performing ELISA on IP samples.

Is there an IP elution buffer that I could use that is compatible with ELISA?

Thanks

I am conducting a co-immunoprecipitation experiment and when running a random rabbit IgG negative control, my protein of interest binds highly to it. Not quite as much my protein specific antibody but still quite high. I have reduced antibody concentration and increased the % tween in the wash buffers but it doesn't seem to help too much. Any suggestions?

Greetings

I am looking for a recommendation for a generalistic E. coli antibody (i.e. an antibody that recognize many strains of E. coli) to be used in immunoprecipitation experiments (i.e. it need to recognize efficiently whole cells.

Many thanks

Yoram

Hi,

I am planning to tag a gene endogenously (in vivo) in E. coli for immunoprecipitation. Can anyone suggest an elegant protocol for this? Thanks!

Hi everyone!

Im performing some supernatant IPs after serum starvation treatment to analyze whether my protein of interest is secreted or not. Since some cells might die during procedure, I can’t distinguish whether my WB signal is due to my protein being secreted or, on the other hand, due to dead cells which cytoplasmic content is released to the culture media.

Thanks in advance.

I am trying to purify secreted FLAG tag protein in the cell culture supernatant using FLAG Immunoprecipitation Kit. I am wonder should I add protease cocktail inhibitor in the supernatant for preventing the protein from degradation?

Any suggestion? Please help me.

Thanks a lot.

I am a doing DNA pull down assay using streptavidin couples agarose beads ( Sigma). I have two biotin labeled ds DNA probes - wild type and mutant ones. I have prepared a matermix using binding buffer, poly DI-DC , 100ug nuclear extract and DNA probe, incubated for 30mins at 25⁰C and then added 40ul of streptavidin beads into this mixture and rotated for 2hrs at 4⁰C. After that I washed the beads for 2times using the binding buffer and eluted by 40ul of 5X laemmelli sample buffer. I have separated 50ug of nuclear extract previously as an input control, added 40ul of LB and used it as a positive control in western blot. In western blot, I have noticed that, the intensity of the input sample is always less in comparison to the wild type probe whereas there is a significant intensity difference between wild type vs. mutant probe. I can not understand whether this result is correct or not. Generally the input intensity should be more than pull down fraction but in my case this is opposite. Please give me some valuable advices so that I could rectify the problem. The image is attached below.

Thank you

I was doing immunoprecipitation with protein A/G plus sepharose. After incubating the beads with cell lysates, I washed the beads for 4 times and then add sample buffer directly onto the beads, boil at 95.2 °C for 5 min, and load this sample directly to the gel. Normally I use 2x sample buffer for this, but today I accidentally added 5x sample buffer onto the beads. I boiled the sample at 95.2 °C for 5 min as well. What will happen if I load this sample to the gel? Will it run normally? Also, can I load this sample side-by-side with IPs that were eluted into 2x sample buffer on the gel?--will the samples diffuse to nearby lanes due to the different concentration of components? I don't want to dilute my sample as the protein concentration is not high...

Thank you! !

PIWIL4，piwi-like 4 。

Hi,

Hello everyone,

I am working to isolate/immunoprecipitate homologs of a protein (RdR) from an invertebrate. There are no commercially available antibody for any of the homologs for my organism. However, there exist a polyclonal antibody against another homolog of the protein in C. elegans. Since the two proteins are related and predicted to have similar function (this have not been experimentally verified), is it possible that I can obtain the said polyclonal antibody and use it to immunoprecipitate my protein of interest? and what things do I need to consider if I should/shouldn't go this route?

Is there anything you guys suggest I do?

Also, I'd like to point out that the predicted targets (epitops) by the company are not a 100% match to the homologs I am working with as seen below from multiple sequence alignment.

If the question isn't clear enough I can explain more. Thanks in advance

I'm currently purifying a protein complex from crude cell lysate using immunoaffinity purification. I've screened a monoclonal antibody against a peptide epitope with 5nM binding affinity. I coupled antibody to CNBr-Activated Sepharose 4B resin for IP. After incubation with crude cell lysate, we found that target protein absorbed to the resin using both silver staining and western blot. However, we can't elute our target protein even using very high concentration of epitope peptide (10mg/ml). What might be the reason?

Hi there,

For my IP experiments i'd like to purchase Dynabeads™ Protein A and all that i need for immunoprecipitation. Ive seen they sell kits and you can even buy it individually. What is weird to me, is the fact that they dont sell the "Antibody (Ab) Washing and Binding buffer", and "Washing Buffer" individually. (https://www.thermofisher.com/order/catalog/product/10018D?SID=srch-srp-10018D#/10018D?SID=srch-srp-10018D)

So my question is:

What do you use for washing antybody binding and the washing steps?

Is PBS (+/-BSA) a good alternative for the binding step and PBS+0.01%Tween for washing?

Thank you in advance!

Hello everyone!

I want to immunoprecipitate a protein and then, performe Western Blotting agains phospho-Serin. I would like to know if you could recommend me a good anti-pSer you know is working properly.

I have already try one and it is not.. so before buying a different one I would like, please, your experiences!