- Steffen Larsen added an answer:2How much proteinase-K should one take to overcome common protease inhibitor cocktails?
I am performing an assay inspired by the PIP-seq approach which is essentially a nuclease protection assay. Shortly, the process entails: 1) Cross-linking of RNA with RNA-binding proteins. 2) Lysis of cells and nuclease treatment of lysate. 3) Cross-link reversal (proteinase K plus heat) and purification of protected RNA fragments.
I have an issue with their approach: They use RIP buffer for lysis and downstream treatments and their buffer contains a protease inhibitor cocktail (probably cOmplete Mini tablets), which inhibits proteinase-K and other serine proteases. Yet, when they reverse the crosslinks, they initially add proteinase K to a final concentration of 1 mg/mL.
Do any of you know if this concentration is enough to overcome the added protease inhibitors? I believe the proteinase K activity is central at this point as otherwise the nuclease would still be active when the crosslinks are reversed, which could lead to digestion of the RNA targets of interest.
I contacted MercK, but they would not tell me the concentration of serine protease inhibitor in a standard mixture (1 mini tablet per 10 mL), so I would be grateful if any of you have experience in this matter. Thanks in advance.
Thanks, Brian. Just the answer I was looking for:-)Following
- Kunal Dayma added an answer:9Protein binding too tightly to the beads.I have been trying to elute protein of my interest tagged to GST using a protocol containing 10mM reduced glutathione buffered at pH 8.0. However, contrary to the proposed protocol for this elution to take place, I am having much less success as more than 95% of the fused protein is remaining bound to the beads (beads for binding to GST). I even tried to supplement the protocol by adding 2-mercaptoethanol so that glutathione would be kept at the reduced form. Still, the protein is not getting eluted. Can anyone please suggest the reason for this and any modifications that need to be done for proper elution from the beads?
i have just eluted my protein from GST beads today
I have used 5mM GST but the pH of tris buffer was 9.
I think my elution has been around 70%
so, may be you can increase the pH to 9Following
- Jeshle Vijayakumar added an answer:4How do I get rid of the nucleotides bound to an RNA binding protein?
I am working with an RNA binding protein. The protein is in 1M KCl, 50mM tris and 5% glycerol. I do MBP purification, Dialysis and gel filtration before i finally concentrate the protein. I need this protein for RNA assay and hence i can't use RNAse (or is there a way to use and efficiently get rid of the RNAse? ) and this protein at low salts form aggregats. Any suggestions on how to get a diffuse nucleotide free protein, please?
Thank you for all the suggestions everybody :) At the moment i have decided on adding RNAse in the lysis buffer as suggested by antonio ariza and track the peak during the size exclusion. Hopefully things go well!!Following
- Matthias R. Schaefer added an answer:3RNA binding protein immunoprecipitation (RIP) - Can someone help with problems with nuclear lysis of lymphoma cell lines?
I am trying for some time now to adjust an RIP protocol for analysis of nuclear RNA binding proteins. The protocol is already applied in our lab for Ago2 IP in order to identify microRNA targets, but Ago2 is of course mainly located in the cytoplasm.
I am trying to apply the protocol to isolated nuclei in order to study RNA interactions with epigenetic complexes. However, I cannot get the contents of the nucleus into solution. Absurldy, very similar protocols are applied by multiple groups to the exact same end, but it does not work in my hands. I was wondering if this may be a cell type specific problem, as I work with suspension cells (B cell lymphoma cell lines) and the protocol is usually applied to adhesive cells.
Protocol (lysis part) in brief is as follows:
- Isolate nuclei
- Resuspend nuclei pellet in 200uL cold polysome lysis buffer (10mM HEPES pH7, 100mM KCl, 5mM MgCl2, 0,5% NP-40, 1mM DTT, RNAse and protease inhibitors) and lyse on ice for 30min, vortexing at regular intervals
- Clear lysate by centrifugation (14.000g 10min 4C)
- Use supernatant as input for RIP
Unfortunately, my protein and RNA is all in the waste pellet instead of the supernatant. I have already tried multiple things to assist nuclear disruption including sonicaton, dounce homogenization, bead beating, addition of enzymes such as DNAse as well as combinations thereof. I do not want to play with the salt concentrations in the buffer too much as this may disrupt the interaction I am looking for.
So my questions are:
- Is anyone aware of additional tricks (disruption methods, buffer additions) for nuclear lysis?
- Is this a problem stemming from working with suspension cells?
- Is anyone aware of protocols for (R)IPs that are optimized for suspension cells?
- Is there another way to pre-clear my lysate? (centrifugation causes me to lose not only debris but also everything I am interested in)
This is not a cell type-specific but a general problem. I guess that your nuclear extraction buffer is too mild. You need at least 0.5% deoxycholate and maybe minimal amounts of SDS (0.05%) in it.
- Melanie Winkle added an answer:2Any efficient assay to determine unknown protein that binds to a known RNA sequence?
I am looking for an assay that can identify the protein that binds to a RNA sequence I am interested in. I have found many assays that accomplish the opposite; in which they can determine a know protein's target RNA sequence. The RNA sequence of interest in an extended 3' UTR of a RNA transcript in the axon of a mammalian neuron.
Chromatin Isolation by RNA purification (ChIRP)
This is essentially a pulldown of RNA using tiling biotinylated oligonucleotides against your RNA of interest. You can then analyze the DNA and proteins bound to it by sequencing and mass spectrometry. This method needs a high input though, not sure if applicable to your cell type.
Otherwise maybe an EMSA with a cell extract. You will know if something interacts with your RNA, I am not sure about how to purify and analyze proteins after an EMSA though.Following
- Anoumid Vaziri added an answer:4How can I detect an unknown RNA, after performing RIP?
I am trying to pull down the RNA molecules bound to my native protein of interest in the plant Brassica rapa (close to Arabidopsis) by RNA binding protein immunoprecipitation, but I have no idea what RNA molecule I am trying to detect, therefore I have no way of designing primers for subsequent detection. Are there any methods that I could try that do not involve RNAseq? I'm familiar with the RAGE technique (cutting up cDNA and polyadenylating them to bind to adapters) but I wanted to know if there are any other techniques that I could use?
Thank you for your advice, I'm definitely considering a cDNA library based method but in the sense that, I create cDNA then digest it with blunt end cutters and subsequently polyadenylate them. Then I'm thinking I can apply a rapid amplification based technique that can ligate adapters to the poly A tail and go from there.Following
- Sue yu added an answer:7What kind of method can be used to test a potential RNA-binding protein can bind to RNA?
The protein I am studying has a domain may related to RNA-binding, but so far no paper showed it could bind to RNA.
I want to test whether it can bind to RNA directly, what kind of methods can be used under the situation that I do not know the potential RNA targets?
Thank you for all the answers. They are really helpful.Following
- Dmitry S Karpov added an answer:13How could I check whether there is RNA in my protein solution?
I purified a recombinant RNA binding protein from E.coli, I worried that my RNA-binding protein was contaminated with host RNA during purification. I need to remove RNA completely, I have treated with RNase A and use 1M salt in the buffer, and How can I check whether there is still RNA in the protein solution during purification? such as gel electrophoresis or NanoDrop based on 280/260? Thanks a lot.
After treatment with RNAse, the RNA fraction bound to your protein and thus protected from RNAse may be so small that could not be reliably detected spectrophotometrically at 260/280 using NanoDrop. Try much sensitive Qubit fluorometer with RNA specific fluorescent dyes. Qubit measures picograms while Nanodrop from 5 nanogramm of nucleic acids in microliter.Following
- Zixun Han added an answer:4How can I remove bound RNA of RNA binding protein during purification?
I have a problem for several months. I desperately need your help.
I have two tagged proteins GST-A and His-B, both of them are RNA binding protein, pI of GST-A is 6, pI of His-B is 9. Previous reports says A and B has interaction, but in the presence of RNA, the interaction will be disrupted.
My aim is to purify this A-B complex, and try to determine the crystal structure.
During my purification, after sonication, I treated with 0.2 mg/mL RNase A in 35℃ for 30 min. Then the tagged A and B protein were purified by Ni-NTA or GST beads with buffer containg 1.5 M NaCl, after removing the tags, desalting to 100 mM NaCl, mix in ratio, and load on gel filtration/size exclusion chromatography with 100 mM NaCl, there is completely no binding between A and B, and I found there is still RNA in the peak based on UV 280/260.
If so, how can I completely remove RNA? Thanks a lot.
Thanks for Gavin, Dominique and Utkars' answers, I will follow your suggestion and try once again.Following
- Archana D. Siddam added an answer:8Whats the best way to determine what is binding to the mRNA 3' UTR and blocking a miRNA from binding to it?
I have two cell lines, in one the miRNA binds to its target mRNA, in the other it seems to be block. How do you determine what is blocking the miRNA from binding to the mRNA? Is it protein or RNA? What is the best way to get at the answer? Thanks! -j
I agree with Kyle. Here is anoher recent Natute paper talking about a ncRNAs/circular RNA acting as a sponge for a particulate miRNA in Ago dependent manner. This paper should provide more clues to your hypothesis. Like any other RNA binding protein, AGO protein complex is conserved, so there might be a possibility of your cell line expressing this particular ncRNA. You can quickly do a qRT-PCR to determine its expression or any other ncRNA that you identified using the above database in your cell line and go from there. Good luck!
- Hanh Nguyen added an answer:3Is 1500 nucleotides RNA too big to do a RNA -protein binding gel shift?
It is the first time I do a gel shift for RNA. I feel kind of lost. I need to do several RNA protein binding gel shifts. My purify protein is 10 KDa and my RNAs is 200, 600, 1500, 1600 nucleotides. Do you think I can see the shift on the gel without using radioactive? Are those RNA sizes too big for RNA gel shift? I really appreciate any responses from you.
Thank you very much!
Dear Craig A Gelfand and Prescott Deininger ,
Thank you very much for your responses. Those help me a lot. I'll cut my sequences down to around 200 nucleotides instead. I hope this should work. I forgot to mention both of my protein and RNAs are purified ones. In the active form, the protein stays in hexamer form around 60 Kda. I'll labelled my rna with biotin for Lishtshift chemiluminescent. That case I can switch to a pool down if it is necessary.
- Arjen Boender added an answer:6Any experts on post-transcriptional modulation of mRNAs?
I am interested in the expressional regulation of an mRNA that has three different isoforms: one with almost no 3'-UTR and two with 3'-UTRs of about 6000 nucleotides. I am wondering what I can deduce from this fact? For example, is it possible that the mRNA lacking the 3'-UTR is constitutively expressed, while the others are under regulation of post-transcriptional mechanisms? If anybody knows some good reviews about this topic, please share!
Thanks Francesco! If am working in glial cells, but I think this should also go for glial mRNAsFollowing
- Man Liu added an answer:14How can I reduce non-specific binding of proteins on my streptavidin beads?
Dear all, I'm doing RNA pulldown experiments to identify RNA-binding proteins which interact with my RNA. I biotinylated my RNA using biotin RNA labeling mix (roche) and PAGE purified the RNA. For binding, I overnight incubated 5 ug (~80 pmoles) of biotinylated RNA with 1.6 mg of Hela nuclear extract in the presence of 0.1 ug/ul tRNA and 1U/ul RNasin. For pulldown, I added 30 ul of streptavidin agarose beads (Thermo) to the binding reaction and incubated for 1 hr. After extensitve washing, I eluted captured proteins in SDS sample buffer and performed SDS-PAGE. Following coomassie staining, I could observe several bands specifically enriched in the RNA(+) sample, but the problem is that there are too many non-specific proteins appearing in both beads-only and RNA(+) samples to precisely excise out bands of interest without contamination. I tried pre-clearing my extract by incubating it with 60 ul of streptavidin beads before binding, but this did not significantly improve specificity. Can anybody help me reduce non-specific protein binding on beads? Any comments would be greatly appreciated.
I encounter the same problem as you did and I'm wondering whether you sovled it or not and how. Thanks a lot!Following
- Ag Muhammad Sagaf Abu Bakar added an answer:2Has anyone expressed a recombinant double stranded RNA binding protein?
I am trying to clone the mentioned gene into a pET vector, preferably with no fusion partner. Each time I try, I get no transformants. Could this gene be toxic to the host?
Thanks for your input. I am transforming the ligation mixture into TOP10 E. coli (DH5-alpha strain), as directly transforming it into a BL21 might cause basal expression and can cause toxicity.Following
- Christopher Xiaoquan Sun added an answer:2Are you familiar with RNA-Protein docking?
I would like to docking the LNA (locked nucleic acid) with a protein so that the ligand (LNA) should be flexible. Which docking server or software can help me for this job? The LNA is about 80mer. Best regards.
Autodock is also fine. You can find video tutorial on Youtube and many many relative literatures.Following
- Brent Passer added an answer:4Where do I start screening for RNA-protein interactions?
I want to identify the host proteins that interact with the non-coding RNA of virus. Is it possible to do the screening on the similar lines as done in protein-protein interactions using Y2H.
We offer RNA-protein screening platform at Hybrigenics. Here is the link.
- Arjen Boender added an answer:3How do I determine which specific RNAs are bound to a RNA-binding protein at different timepoints?
I just want to know if it possible to profile differences in the type and amounts of RNA that RNA-binding proteins bind during a behavioral task. I expect that this would change during different learning stages, but I would like to test if this is really true. I know about CLIP-IP etc, but I was wondering if it is possible with these methods to compare the amount of binding of a specific mRNA to an RNA-binding protein throughout different learning stages and for example say that mRNA A is more bound to protein in B in the later stages of learning
- Shuba Varshini Alampalli added an answer:7What is the best strategy to catch a regulatory non-coding RNA in action ?
I am a studying a long ncRNA in S. aureus. This ncRNA is important for virulence but no molecular mechanism or interaction studies have been performed yet. This could have possible post-transcriptional control in virulence regulation.
I want to know what other possible RNA species or even proteins, does this ncRNA interact with or regulates. For this I need a good strategy to investigate interacting RNAs or proteins from the bacterium ( would be nice to know it on a genome-wide scale)
What are the constraints?
Do I need an overexpression ? do I have to used non-chaotropic conditions and what are the associated points to be considered?
I would be much obliged in someone could help me out with designing a strategy.
Thank you Sudip for putting up this query. I was also having trouble finding ncRNA targets in malaria parasite. And now I have few strategies to dwell on.Following
- Gabrielle Haas added an answer:3How to design the RNA oligos for RNA/Protein pull down assay?
I have to do a Biotinylated RNA pull-down assay to verify the ability of two RNA binding proteins to bind RNA of interest. I’m working on designing the oligos. I wonder it is better to add biotin at 3’ end or at 5’end, and whether a sequence spacer is needed between the two binding sites and what composition it should have (random?).
Thanks in advance
In my opinion, the orientation doesn't matter as soon as your choice is not interfering biologically with your system. To explain, in my case my bait was carrying the biotin in 3' because I wanted to reduce the steric hindrance with the 3' of my prey. So, I made a choice knowing it would be the best orientation.
So I guess that if you don't know if the orientation might influence or not, maybe try both separately. It might be an option. In any case, I would highly recommend a spacer between the oligo and the biotin.Following
- Marco Mazzorana added an answer:4How can I inhibit prokaryotic RNases?
I would like to use biotin-RNA to fish RNA binding proteins from E. coli extracts. Do you have any idea how to prevent RNases from chopping off RNA?
If metal-binding is not required for the interaction between your protein/s and RNA, I assume you can just use EDTA to chelate the catalytic Mg2+ in RNAse thereby blocking the enzyme activity.
Biotin/Streptavidin bindind is not affected by EDTA, as far as I know.
- Pradip Kumar Biswas added an answer:4How can I remove RNase A from my Ni column-purified protein?
My protein is an RNA binding protein which necessitates removal of RNA for its activity.
I assumed by affinity chromatography that RNase A would be removed as it is an affinity-based separation, but I still have residual RNase A activity which is hurting my assay.
Dear Adam, Paco and Anna thank you so much for all the suggestions!!!! I did pass my crude extract on DEAE in presence of 200mM NaCl as I know my protein will not bind to DEAE at that [NaCl] while the RNA and DNA should bind to the resin. The flow through was charged on Ni column and am yet to see the results. Thanks for all the suggestions. I will try out Anna's way if things do not work out. Thanks a ton guysFollowing
- Maria Letizia Di Martino added an answer:4In which volume should I dissolve tRNA-fMet from Sigma (10U) to obtain a 10uM solution?
Hi! I will perform toeprint assays. I got the tRNA-fMet from Sigma (10U). The only indication from sigma is that 1U will have acceptor activity of 1000 pmoles /A260 unit. In my protocol I should use 10 uM solution of tRNA-fMet, but no MW is indicated. Can anyone help me?
Ok...è il calcolo che avevo fatto. Il dubbio era che le pmoli sono riferite all' attività e non alla concentrazione. GrazieFollowing
- Maria Wiktoria Górna added an answer:3Why am I seeing a Subshift in the RNA-protein after running EMSA?
I've run a number of RNA-protein and RNA-peptide EMSAs but this is my first sub-shift (complex running below free) result. I'm wondering if others have seen before? Google and literature have been little help and my labmates/PI are a bit shocked also. Is this suggesting a major structure change upon binding? (this would be cool) Should I be more worried about aggregation?
I've ruled out RNAse contamination after running denaturing gel of RNA-Protein complex, also have 3 controls of different binding and non-binding RNAs of same size on same gel with same protein sample added. The RNA showing the sub-shift is a 34mer stem loop with 2x1 internal loop and 18nt single strand region. The protein is 80AA with single RNA binding site.
Maybe run DLS to confirm not aggregation related problem? Maybe fluorecence anisotropy? Or DOSY-NMR?
I've tried different gel %'s (from 8-15%), different buffering systems, different bis/acrylamide ratios, adding detergents, tRNAs, different running temps, and voltages, gel thickness. All have helped sharpen the bands but the subshift is still there.
- beulah mae ann villalobos Solivio added an answer:3What is the best program for determining the minimization energy of a protein structure bound to a nucleotide?
I am trying to find the minimization energy of an RNA binding protein when bound to a specific nucleotide. I tried CHARMMING but it seems like it only allows protein-protein interaction. So far, Arguslab worked for me but I'm hoping to try other programs, too. Any suggestions will be really helpful. Thank you!
Thank you so much. This really helpsFollowing
- Alexander Rohe added an answer:5How sensitive is FITC to heat and pH changes for the purposes of fluorescence anisotropy?I have recently tried using fluorescence anisotropy to measure FITC-tagged shRNA binding to an RNA binding protein. My questions are:
1.) Will running an annealing program with high temperatures to properly fold the RNA will cause the FITC tag to dissociate from the RNA?
Annealing program :
95C 3 min
Ramp cool -0.1% 10 sec
repeat 700X (brings temp to 25C)
Hold 25C 5 min
4C hold forever
This particular program takes about 2 hours to complete.
2.) Is the buffer type and pH very important for making sure FITC stays "happy"?
Our buffer we are using is a Phophate based buffer containging: 20 mM NaPO4, 50 mM NaCl, 1mM EDTA pH6.8.
Should we change the buffer base to something like HEPES or Tris?
Our experiment parameters were a stable 10 nM shRNA and variable protein concentrations:
.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1000 nM
Any help would be greatly appreciated! We just aren't sure why we are not seeing binding of the RNA to the protein which has been shown to bind to this protein in published studies.
Just a general note towards fluorescence anisotropy: Sinve it is quantified via the emission ratio of both measured intensities (parallel and perpendicular to the exciting plane), fluorescence anisotropy does not depend on the overall fluorescence intensity.
Therefore, as along as your total fluorescence intensity is high enough to ensure an adequate measurement error, your anisotropy results won't be negatively influenced by photobleaching.
However, since anisotropy is an additive entity, fluorescein-derivatives derived from your labelled shRNA (e.g. breaking of the covalent linker) will definitely interfere with your measurements by narrowing the assay window. Depending on the extent of "free" fluorophore, it may even cause an assay window of zero...Following
- Sneha Singh added an answer:1How do you design a primer for an RNA target?We are trying to develop a biosensor to detect the Dengue virus (DENV) in field condition. Since DENV is an RNA virus, I need to design primers that can specifically bind to RNA. These primers are not for the purpose of any form of PCR but only to capture DENV.Hi Shishir.............. I believe you can use the same primers as we use for RT PCR to detect RNA in the field since DENV RNA lacks a poly A tail, we use its specific reverse primer for preparing the cDNA. You can take help from Anoop M et al., 2010; 2012.Following
- Steffan Noe Christiansen added an answer:3Does anyone have any suggestions on how to solve some problem with EMSA for Detecting RNA-Protein complexes?I am having problem with my proteins that have ~10 and ~11 pI and 17kD and 14kDa respectively. They do not get into the gel because of their charge. I tried purifying them even with a maltose binding protein tag to reduce the pI, however they are still too basic to migrate into the gel. My RNA is 130 nt long. I see the unbound RNA and decreasing concentration of unbound RNA with increasing protein concentration but the bands corresponding to RNA-protein complex are not very clear. Proteins alone are not showing up at all. I am using 5% Native PAGE. I am staining the RNA with Sybr Green and Protein with Sypro Red. Since I don't see clear complex bands, I cannot estimate the Kd. Any ideas on how to improve the quality of the bands and how to get the proteins to migrate into the gel instead of moving backwards? I tried non-ionic detergents such as Triton etc. but this did not help.
I am in a similar situation trying to estimate Kd values for a basic protein (pI = 9.8) and its binding to different sequences (25-30 bp). Did you ever solve your problem how to design your EMSA, so you were able to determine Kd values? At the moment, I am only able to show a low extent of binding between the protein and the sequences even though the interaction between the protein and the sequences earlier have been shown to be in the order of ~5-80 nM.Following
- Emmanuel Po-Shun Wang added an answer:16What's the best way to prove direct binding between protein(s) and RNA?How to prove PROTEIN-RNA binding is direct or indirect?RIP assays can be performed using the EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit.Following
- Deepak Kumar asked a question:OpenProtein-RNA simulation - can anyone help?I am looking to do :
a) free energy calculations (linear interaction energy) in order to analyze the contributions of the protein residues to the binding of RNA.
b) analyze the specificity by running computational alanine scanning.
Can anyone tell me what simulation tool/software could be used to accomplish these tasks?Following