- Jovencio Hilario added an answer:Can you recommend useful protocols for removing bound DNA that co-purifies with recombinant proteins?DNA enzymes and binding proteins expressed in E. coli often have bound DNA or RNA that can be troublesome to remove completely for biochemical experiments and crystallizations.
If your protein constructs have tags (GST or His), you can try on-column washing with 1 M NaCl. I utilize this successfully when purifying certain recombination proteins. Also, a high-resolution ion exchange should work as well at the final step of purification.. Either Bio-Rad ENrich Q/S or GE Mono Q/S.Following
- Chasper Puorger added an answer:Can someone suggest a way to elute biotinylated proteins from avidin agarose beads?
Generally in all the elution protocols, the proteins are eluted in very harsh conditions. E.g Boiling in SDS sample buffer, elution by using Guanidine HCl. This creates a lot of avidin background when run on a gel. I have to submit my sample for LC MS. In order to do that I need to reduce the noise. Can anyone suggest me any method that can reduce the background noise?
I had good results by eluting peptides from streptavidin beads with H2O at 80 °C (5-10 minutes incubation). I analyzed the samples with LC-MS and obtained good results. Elution with SDS or GdmCl resulted in huge background.Following
- Tran Nguyen added an answer:Anyone can help me with the SEC-HPLC analysis of protein interaction between LdPEX5(203-391) and LdPEX14(1-120)?
Thanks in advance for any help.
Thanks a lot for your help. :) I'm getting through it..Following
- TEV protease precipitating? I am purifying a GST-tagged protein, and cleaving the tag with TEV protease. My buffer contains 20 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM DTT. There is 75 mM reduced glutathione around from elution, and I add 0.5 mM EDTA for digestion. I incubated overnight (ratio 1 mg enzyme to 10 mg protein) at 4 degrees.
In the morning, the solution was cloudy. So I spun out the precipitate, and took samples to analyze by SDS-PAGE. Some of my protein precipitated, but most (90%) stayed in solution. The 27 kDa TEV protease precipitated, and this was evident because most of my fusion protein was not cleaved. Another lab member used the same prep of protease (different protein), and had no problems.
Anyone have any ideas as to what is going on here? Has anyone ever heard of TEV protease precipitating?
It could depend on the TEV solubility mutations. Try this one: http://www.protean.cz/en/recombinant-protein/28/tev-proteaseFollowing
- What is the best method for separating and isolating a purified protein from the His-Tag in a solution? We purified a protein marked with His-Tag and with a TEV cleavage site. We would like to know ideas about how we can separate the mix of proteins in the solution after the addition of TEV protease.
A good source could be found here:Following
- WHy is my protein gone after TEV digestion?
Can you please suggest to me some comments relating to this trouble.
I did the protein expression and purification from E.coli cells. In this case, I used the MBP tag and I also cloned the TEV cutting site to remove the MBP tag after purification.
However, after I treated the fusion protein with TEV, I could not get my target protein even there is no TEV cutting site inside of my interested protein.
Can you please advice me!
Thank you very much! ^^
The problem could be with the TEV protease impurities. Try this one:Following
- Kristine Atkinson added an answer:Can a phosphorylated protein be separated into phosphorylation or non-phosphorylation band in phos-tag PAGE and have a range of molecular weights?
A protein that be phosphorylated should be separated into phosphorylation or non-phosphorylation band in Phos-tag SDS-PAGE.However,does there exist a range of molecular weight of the protein to make sure successfully detect the two forms? Based on your own experience or expeiment.
That is a complicated paper, Xianfeng-- and it focuses on the ER, so are you sure the extraction procedure you used was correct for your own protein? There were other worrisome parts of the procedure, such as phosphatase reversal-- not enzymes I'd wish to have floating around the lab. Perhaps this earlier Kinoshita paper is useful, if you're married to the idea of using molecular tags-- http://www.ncbi.nlm.nih.gov/pubmed/16340016/
HRP-labeled tags always require wisdom in analysis, the enzyme can have unwelcome chemical consequences (I prefer immunogold). An isoelectric focusing method would exploit the change in pI as a consequence of phosphorylation-- for a review of many types of post-translational modifications that exhibit a pI shift see: http://nar.oxfordjournals.org/content/32/suppl_2/W638.fullFollowing
- Subin Cheri Kunnumal Raj added an answer:Could anyone help me in finding a method to extract a protein extract with high protein concentration?
l have tried to prepare a proteina protein extract with high protein concentration through different ways but I did not succeed. maximum content of protein is 30% whereas I expected around 70 to 90%. I would be grateful if anyone have experience in this area could help me.
The extract you obtained could be concentrated by rotary evaporator or lyophilizer. So you would obtain a concentrated solution of protein. I am assuming that you have isolated the protein of interest. If you could elaborate a bit more on your problem, I could be a bit more clear.Following
- Edward Michelini added an answer:Why do I see precipitations during purification using Nickel resins?
I'm working with a recombinant protein with size of 75kDa. It contains His-tag which facilitates with downstream purification process. I used in vitro refolding techniques to scale-up production of this protein. So far I was able to obtain fairly large volume of soluble, refolded protein. However, when I tried to purify the protein using nickel resins, I see precipitation. Unlike the traditional affinity column, I'm mixing nickel resins directly with my protein solution in cold room for period of 8 hours. After 8 hours, what was initially a clear protein solution is now filled with precipitates. Please let me know if you have any suggestions on how I should purify this His-tagged protein and also provide possible explanations for accumulation of precipitates. Thanks!
Batch absorption for IMAC can work well but several things can happen that contribute to protein aggregation, including proteolysis, metal exchange with the resin and/or oxidation of the proteins. Try flow binding by recirculating the extract and pull samples at various times to follow binding. One or two hours often sees all the binding thats going to happen. Addition of 1-5mM TCEP will guard against oxidation induced aggregation and a protease inhibitor cocktail (no EDTA!) will stop degradation.
You can also check if precipitate is your protein, has different metals etc.Following
- Abbas Raza added an answer:Has anyone come across S-myc tag rather than the conventional c-myc protein tag? Can we detect this protein tag using anti-c-myc antibodies or not?
Has anyone come across using S-myc tag rather than the conventional c-myc protein tag? I received a construct that had S-myc and I am not sure if this is the same tag as c-myc. There is no homology as far as I can see in the two tags. If anyone has used this tag or can guide me about it like can we detect the protein using anti-c-myc antibodies or not?
thanks Mark, i have the problem sorted. the person who had provided me the construct had included a signal sequence and labeled it S-myc.Following
- Jan Piwowarski added an answer:How can I improve in vitro translation and protein purification for mRNA display?
I have tried to purify my protein after the in vitro translation using his tag (or poly dT followed by his tag). In the either case I can detect activity after the purification, but I can not see the band on the gel (I tried: Coomassie, silver and fluorescence, once that fluorescein is attached to the RNA), I didn't make a Western blot yet. Does anyone know some tricks to increase the in vitro expression and obtain greater amount of protein after purification steps? I'm using Rabbit Reticulocyte Lysate System.
The mRNA/proteins are someties sticking to the eppendorf tubes depending of the platic used for its production. Try low-binding or siliconised one.
Regards - JanFollowing
- Anthony Partridge added an answer:Can anyone suggest other tags for purifying proteins under denaturing conditions other than Histidine-Tagged?
We would like to find an option to purify proteins under denaturing conditions (= 6M guanidinium hydrochloride) that is not his column (or similar, cobalt etc). Any ideas?
Thanks a lot!
Since you are denaturing your protein anyways, use reverse phase HPLC. If necessary, a preperative column can be used for larger scale. This has been applied in the past to purify proteins that have been denatured from inclusion bodies. Look up publications that use KSI fusions (e.g. http://pubs.acs.org/doi/full/10.1021/bi800107a). Agree that you should do the inclusion body purification as a first step.Following
- Bo Pontoppidan added an answer:How should I set up my FPLC for size exclusion?
I work in a lab with access to the NGC FPLC from biorad and the duo-flow. I want to set up a protocol for size exclusion and desalting. I was wondering if anyone has helpful tips for the set up. I want to buy the resin and reuse it for separating proteins in the range of 11-130 kDa. I have the MT-5 and MT-10 columns from Biorad as well.
What resin should I get that would be compatible with columns that have a 5-10 cm length.
For separation of proteins from each other (high resolution gel filtration) and for separation of proteins from salts (group separation) you need different columns. Different media in the column and different length. Read the book Jiri linked to above and you will have all your answers.
P.S. FPLC is a brand of GE, so what you have from BioRad is not an FPLC :)Following
- David M Jameson added an answer:Can tyrosine intrinsic fluorescence be detected at an emission wavelength either than at 307nm?
I'm doing fluorescence spectroscopy on my protein expecting to see the intrinsic fluorescence of tyrosine and also its effect with addition of cu(i). The excitation wavelength was 280 nm while the emission wavelength were from 300-500nm. From my reading, it supposed that tyrosine will have an emission peak at 307nm. However, in my case, an emission peak appeared at 380nm. None at 307nm. What can that be? Is it still tyrosine or something else? My protein is in MOPS buffer pH 7.
Its conceivable that you are seeing emission from tyrosinate, although that should be in the 350 - 360nm range. I presume that you have no tryptophans in your protein (otherwise you would not expect to observe the tyrosine emission). Which spectrofluorimeter are you using? Possible you are seeing an instrumental artifact. Is you emission broad and smooth - what wavelength range does it cover?Following
- Pegah Saremirad added an answer:Why would a size exclusion column not show reproducible results?
Anyone have an idea about the reproducibility in size exclusion column, silanol silica type. I am doing my experiment to study enzyme- substrate interaction; a negative peak is observed but the intensity of the peak varies, what could be the reason?
Hello acetone and blue dextran are two different tests. I would strat with acetone test as blue destran sometimes tend to get stuck on SEC matrix. Inject 2-5% (v/v) acetone in water/buffer on your column and elute with water/buffer. I used water as acetone did not interact with the matrix. If the peak is symmetric that's a good sign. If you see peak tailing the column is not clean and that explains back pressure. If the peak is leading (i.e you are detecting part of acetone earlier that the total volume, the column could be over packed and you have channeling in the column which can happen for various reasons.Following
- Tomáš Hluska added an answer:What does resuspension of cell pellet to a density of OD(at 595 nm) = 50 means?
My protein is a Trx fused protein and most recommended method of cell lysis is osmotic shock which allows selective release of soluble proteins. The protocol suggests to resuspend the cells in osmotic shock buffer to the density of OD = 50; when OD value is taken at 595nm. As far as my knowledge goes, OD value cannot be this high. Kindly suggests what it means or an alternative strategy to practically perform this resuspension.
it's exactly as Christian wrote, you need to harvest the cells and then resuspend in such volume to obtain OD 50, not to collect 50 ODs (what is that anyway? You can collect as many cells as you want).
And since the protocol is for osmotic shock, the concentration of cells will be probably crucial.Following
- Leonardo Bianchi added an answer:How can I use information from different protease digests for PTM localization?
it should be possible to enhance your confidence in PTM site localization by combining information from digests with different proteases, e.g. by considering where the peptides overlap and checking if fragment ions that are absent in one case might exist in the other. But that`s easier said than done.. I was wondering if anyone of you has tried this or is aware of any good literature?
1) I would split my sample in two aliquots
2) I would dephosphorylate or deglycosylate one and keep the other one as control
3) I would perform proteolysis compare hydrolysates between treated and untreated aliquots. (repeat for each protease you're willing to use)
for dephosphorylation this protocol was very effective (I used calf intestine alkaline phosphatase from SIGMA, cat P6674):
and for deglycosylation I used the SIGMA Enzymatic protein deglycosylation kit (Cat. EDEGLY) following the product instructions.Following
- Weiping Yang added an answer:How can I increase the binding affinity of GST-tagged protein to GST sepharose column?
I am purifying a protein having GST tag. I am using 20mM tris pH 7.5, 200mM NaCl, 5mMDTT and 5% glycerol as the equlibration buffer. Column is of 2ml bed volume (I have hardly 3mg of my protein). Protein is not completely binding to the column and nearly 30% I can find in the flow through. Please suggest me how to increase the binding capacity of the protein.
Thanks in advance..
Hi I did purification of GST-tagged protein for several times. My protein bound to the resin loosely, so I use more resin, and also I use the PBS buffer pH7.3 ( which was suggested by protocol) instead of Tris. It came out nicely.
The protocol also suggest that you could mix your supernatant of extraction with GST-resin and shake 1 hour at room temperature, then reload to the column.
- Hanifah Adawiyah added an answer:How do I prepare fish scales for collagen extraction?
I'm going to do collagen extraction from fish scales. How to prepare the sample before demineralization process? Is it enough by cutting fish scales into small pieces by using scissor? Or I need disperser to make it become powder?
Would you like telling me about deproteinisation process? I'm not quite understand about it actually. The process is to remove unnecessary protein in the surface, is it? Does collagen will not dissolve in solvent?Following
- Nicola Acerra added an answer:How do I store and prepare beta amyloid solution for spectroscopic experiments?
I am going to procure a beta amyloid protein for spectroscopic experiments please suggest amyloid solution preparation way so that the protein is minimally waste and possible result could be obtain
I agree with the other answers, HFIP is a good organic solvent to store Abeta peptide, but be careful, HFIP is very unstable solution (very "volatile"), working in ice because HFIP evaporate very easily and when you prepare the aliquots the concentration of Abeta peptide could change. However if you looking for a buffer for biophysical experiments of aggregation I suggest you to use Potassium Phosphate (KPP) 10 mM K2HPO4, 10 mM KH2PO4 at pH 7.4. Good luckFollowing
- Tilman Lamparter added an answer:How do you trying out Affinity Chromatography Purification Conditions?
I was doing the His-tagged protein purification by HisTrap FF 5 ml recently using Äkta. I came across a problem that my targeted protein was mainly found in the flowthrough of my column. I tried to ran down the binding buffer imidazole to 5 mM, and stripped and regenerated the column as well. However, the targeted protein were still found in the flowthrough. What methods can I try out? I tried the gradient elution and step elution for the purification condition. For gradient elution, I use 10 x CV, 2 ml/ min flow rate to elute the protein; for step elution I used 20 mM, 50 mM to wash the column first, the targeted protein peak were supposed to come out at around 90 mM (ä which I got from the gradient elution peak position). The good thing of step elution is that some of impure were washed out by the low concentration of imidozale, but there are still quite amount of targeted protein shown in 50 mM band, and the 90 mM peak is as high as those of 50 mM band with the band almost same intensity shown in the gel. If there are any other buffer condition suggestion?
Dear Sisi Xie
we do not use a pump, your 2 ml /min flow rate is certainly correct, I do not see the need to slow down.
Washing: check A 280 nm, should be < 0.01 before elution starts.
Why is there so much in the flow through? You cannot use urea, denaturation is irreversible, we have tried this very hardly.
I believe your column is overloaded have you calculated the mg amount that you load and the capacity of the column?
Batch should work but the purity is not good enough.
I remember there was another message, but I do not see it at present
- Carl Manner added an answer:Can anyone help me to get hold of a pT5 vector ?
I am trying to get hold of a very old school vector for some cloning work we are doing. We are following a protocol and don't want to change ANYTHING, hence the need to get hold of this vector. As far as I can tell from searching it is not commercially available and I have had to go back to patents to find the gene sequence. Rather than having the whole thing synthesised I was wondering if anybody could tell me how/where to get the pT5 vector from?
I haven't found any commercial suppliers, but I did find a patent application from 2009 that uses pT5 (linked below). I would check with the authors and see if they'll send you some empty vector.Following
- Amit Gupta added an answer:How can I remove SDS from my protein lysate for 2DE analysis?
I am following one protocol by that I am trying to take out nucleus proteins but one of my buffer having SDS and in this case I can not use the same sample for 2DE analysis. I tried another detergent on place of SDS but in that case I was not able to dissolve the chromatin protein pellet.
If any of you knows how to remove SDS from protein sample for 2DE analysis, kindly share you view or experience on research gate.
I would like to give further information if neede.
Thanks Dooyoung lee and pavel actually I was aware about some of the protein precipitation methods but was not sure that it will remove the SDS but after reading a informative discussion I use one of the discussed method for removal of SDS.
I will also try to use Amicon if it works fine the I would like to with this.
Thank you so much both of youFollowing
- Saeed Mohammadi added an answer:How can I separate 20 nm carboxylate coated polystyrene beads from 100% Fetal Bovine Serum for size determination?
Dialysis might be problematic because serum contains a myriad of proteins, some of which, such as BSA and IgG, the major constituents of serum no less, have got sizes comparable to the 20 nm nanoparticles I'm trying to isolate. I've tried high speed centrifugation (21000 rcf) and it does pellet the beads, but aspirating the supernatant is rather tricky and repeated washes can possibly cause aggregation.
Could I ask if anyone has any alternative suggestions I could look at?
first of all I don't know which kind of bead did you use?
if you used polystyrene beads, I think you can use a molar strong acid like HCL to denaturation of BSA and then washing the beads using centrifugation. release of carboxylated beads from proteins strongly depends on the pH of the solution.Following
- Shahrooz Ghaderi added an answer:Can I express exogenous proteins in human Jurkat MDA-M231 cell lines ?
I am searching for an approximate value for the number of proteins that are expressed in a human cell line?
Are their any tables / references / reviews for the number of proteins that are expressed by a cell line?
I need a reference value for a bottom-up shotgun proteomic experiment and I want to compare the numbers of protein hits of the MS based experiment with a reference value?
Or are their any suggestions how many proteins are detectable?
I think you must check naturally this gene expresses in your cell line.if your answer is yes your expression system is suitable but if no you must read more about post translation modification of your protein and check again your expression system.Following
- Alexey Atrazhev added an answer:How do I solve my protein purification problem?
I have theoretical PI of my protein from sequence data which is 5.59. I have tried anion exchanger (DEAE-macrosep) starting with buffer at pH 8 and then reducing it down to 6 followed by NaCl elution (250mM-1M). However could not find any activity in the final elution. I further tried a cation exchanger (Macrosep high sulfonated) column starting with pH 4.5 and increasing it upto pH 6.5 followed by elution with 500mM and 1M NaCl. In this case also, I could not find any protein activity in the elutions. I pooled and concentrated the final elution samples while carrying out assay. I am unable to figure out. Before ion-exchange I had partially purified the protein using Ammonium sulphate precipitation. Kindly suggest an alternative. I am not sure of my actual protein size, but it shows activity in retentate when passed through 100KD membrane.
Membrane-based separators such as DEAE-macrosep can bind unspecifically. Proteins re-suspended after ammonium sulfate often get aggregated. I will recommend to try good old DEAE cellulose column instead and run under conditions you tried already with macrosep.Following
- Adam B Shapiro added an answer:Is ATP regenerating system mandatory to use when measuring ATPase activity of a protein?
I am using a pretty old ATPase protocol that measures inorganic phosphate released by The Fiske and Subbarow method( 1925). Nothing is mentioned about ATP regenerationin the protocol. Should I add any ? and if yes, how and when do I add it? I have written the protocal below:
ATPase assay: The reaction mixture in a final volume of 0.25 ml contained 50ul of Tris buffer ( 100mM), 20ul KCl,20ul Mgcl2(125mM), 25uL EDTA( 0.4mM), ATP of 2ul and xug of protein. Incubated at 37 for - hour and reactions topped by adding 50%TCA. Then centrifuged and supernatant was estimated for pi.
0.5 ml of Sup+ 450ul of ammo.molybdate; then 0.4ml ANSA,( made in sodium bisulphate and sodium sulphate)- 15min @37. Blue colour estimated @ 660 nm..moles of inorganic phosphate formed/ ug of protein/hr.
So do I need to add anything as an ATP reg. system?
There are 2 reasons why you might need an ATP regenerating system: (1) depletion of ATP causes the reaction to slow down so that the initial rate of reaction is not maintained, or (2) accumulation of product ADP inhibits the reaction so that the initial rate is not maintained. If neither effect is observed, i.e. if the reaction progress curve is linear for the whole reaction time, then you do not need an ATP regenerating system.Following
- Miroslav Z Papiz added an answer:I want my recombinant protein in soluble fraction.What should I do?
I have a codon optimized construct for a viral gene and I am expressing it in BL21DE3 strain. for all other papers mentioning its purification it is coming in soluble fraction but for me it is coming in inclusion body. I have tried various IPTG concentrations and even various temperatures also, like 37 degree, 18 degree. What should I do to get it in soluble fraction?
I am in agreement with what everyone has said but if you are certain you are following the published protocol to the letter then it must be something subtle that they were doing that they did not report. One thing to consider is the point of harvesting the cells. We have found in one or two cases that even, at 18degC, if the harvesting of cells is delayed by only one or two hours the protein goes from soluble to inclusion bodies. Try different harvesting times. Also one other thing to try is to use autoinduction which does not use IPTG but a glucose/glycerol/lactose mixture so induction is is acieived slowly, this may improve inclusion body problemFollowing
- Pavel Montes de Oca B added an answer:What is the right procedure for VCAM protein detection in TNF-a treated HUVACs cell ?
I am detecting TNF-a treated HUVACs cell's ICAM and VCAM protein expression by western blotting assay. I put cell starvation for 16 hr and drug treatment for 24 hr, using total cell protein for western blotting. Usually ICAM protein is easily detected nevertheless I can't see any VCAM expression. I am wondering is there any other steps have that I need to follow to detect VCAM protein?
Is your Ab agains VCAM working properly? Is it for WB? Normally TNF or IL-1 should induce VCAM in Human umbilical Endothelial Cells.Following
- Tuhin Kumar Guha added an answer:What can I do with the MBP fusion protein expression that does not work?
One of the proteins (homing endonuclease) which I am trying to express and purify has been giving a hard time since.
I have cloned and sequence checked the ORF and its in frame with the MBP (cloned in pMALc5x from NEB), everytime when I try to express this ORF using various IPTG conc., temperatures eyc. it does not. However the empty control pMALc5x expresses very nice when i induce with 0.3 mM IPTG.
The full length protei is never achieved...I am thinking of various pointers like changing the cell strain, doing western, media etc. but I would also appreciate inputs from those who have experienced such problem.
Any suggestion will be appreciated and acknowledged.
Thanks in advance
@Rakesh Kumar : Few years back I tried expressing my protein in a similar manner which you have indicated but did not work with the fusion one though. I will keep your pointers in mind.
@ Pierre Béguin: I guess so. Proteolysis may be the problem. I am trying out various other lower temps. to see whether that makes a difference or not. I have tried his tag but the protein was unable to purify. I appreciate you for the pointers.
@Zeeshan Chaudhry: That will be beyond the scope for the moment, however I thank you for the reply.
@Anna Leopold: Thanks for the reply.I have been doing small scale protein expression to optimize the expression of this fused protein. the prdicetd molecular weight of the fusion protein is 74 Kda (Maltose binding protein is 42 KDa and the recombinant protein is 32 KDa). Ya I agree with you but one time I expressed efficiently 110 KDa yeast peroxisomal protein in E.coli. Well, I tried to purify using his tag but something always goes wrong with that purification.I kinda switched to the pMAL expression system. Western does give the impression that the full length protein is formed at some point and there are few intermediate bands when detected with anti-MBP antibody. pET32 may be a good choice, I can think about that later.
@Hüseyin Besir: Thank you. I tried in vivo endonuclease assay where I co transformed both the expression construct and the substrate plasmid and did in vivo cutting assay. I found the cells were quite viable, so this is not a toxic protein. the sequence of the fusion protein is in frame and we checked it twice and did not observe any mutation whatsoever.
@Ian R Wilkinson: Thanks for the reply. Yes we got me right. The protein is not toxic, the cells are happy even after induction. Its under tight promoter ptac so that's not the problem. Currently, I am transforming my expression construct (in pMAL vector) with pRARE plasmid and hope to see some more full length expression. Time scale experiments and changing media had been done with no such great difference in the expression of the full length fusion protein.
I did not think about the repeat sequences, but we checked the in silico model of the protein, there are no such numerous repeat sequences. pMALp5x can express my fused protein in the periplasm if the protein expression is way too much affected with reducing environment or disulfide bond formation. I have that in mind.
@Christopher Wilson: Thanks for the reply. The insoluble fraction and the soluble fraction kind of look the same. SUMO approach ..lets see you far I can stretch this pMAL stuff.Following