Protein Purification Techniques

Protein Purification Techniques

  • Gary Laco added an answer:
    Any suggestions on affinity chromatography for Aprotinin?

    I am doing affinity chromatography for Aprotinin, I am using trypsin affinity column,equilibration and binding of aprotinin ll be done with Tris HCl buffer pH 8.0 and sodium chloride concentration of 0.3M,I just want to know the role of sodium chloride in this affinity chromatography.

    why sodium chloride is used at such higher molar concentration,for binding?

    if you know any better binding buffer please let me know because i am getting lower binding.

    Gary Laco · The Roskamp Institute

    Adam is right about high salt reducing non-specific interactions with the column.

    Also, aprotinin interacts with the trypsin active site using a Lysine side-chain, so high concentrations of NaCl could disrupt binding of aprotinin to trypsin. Are you sure the protocol called for 0.3 M NaCL? You could try 0.03 M NaCl.

  • Petre Brindusa Alina added an answer:
    How can I analyze by mass spectrometry an intact protein isolated by a pull-down assay?

    Hi to all,

    I'm trying to analyze by mass spec. a DNA Binding protein having a particular conformation, so I'm interested in isolate it and analyze it as intact by mass spectrometry. 

    I've tried to elute the protein with a buffer having 100mM of Glycine at pH 2 and giving a brief boiling (a couple of minutes).

    I know that the elution works because I've assayed it by a western blot.

    The problem is that although a desalting passage, although I eliminated every possible detergent (Tween 20 ......) I always see in my spectra the signal of a PEG-Polymer. 

    Does anyone have any experience?

    Petre Brindusa Alina · Universitatea Alexandru Ioan Cuza

    You need to work on sample preparation ..try a microcon tube with a cut-off depending on the Mw you expect for the protein  and redesolve the protein in a proper buffer for ESI-MS ...or try bottom up or top down MS approach.

  • Prasanta Kumar Ray added an answer:
    What are the most likely reasons for low efficiency of a co-immunoprecipitation?

    I have recently been trying to optimize co-immunoprecipitation in K562 cells. I have tried 6 different antibodies for my protein of interest (all compatible with IP) and am using Protein G-PLUS agarose beads (Santa Cruz) and following their recommended protocol (20ul of beads, 2ug of antibody). I have tried increasing antibody concentrations, increasing bead amount and am still not getting a very good efficiency (ususally lies between 2 and 20 %). I am using cyotosolic fractions in CHAPS buffer and have also tried the C buffer from C/N fractionation kit. What would people think is the most likely next step to try? Is it p[ossible that the beads themselves are the issue and I should try some others?

    Prasanta Kumar Ray · Centre for Applied Science and Technology (CAST)

    First, why you have decided to co-precipitate! If you use one precipitator does not you gt your desired material? Do you get more material for your analysis by coprecipitation? Otherwise,why you have to do that.Think about it.You will have the answer.

  • Niels Dammes added an answer:
    Why do I fail to purify a protein fused to Rat-Fc by protein G?

    Hi everybody,

    I created a recombinant protein consisting of a FLAG tag, my protein of interest and a Rat Fc (IgG2a: Ch1, hinge, Ch2, Ch3). Total size is approximately 60 kDa and the protein is supposed to be soluble and secreted.

    When I try to purify the protein with FPLC by running the cell culture medium over an Akta Hi-Trap protein G column, there is barely any protein in the purified fraction and almost all protein goes straight to the flowthrough. (compared the amount of protein of interest in purified fraction with flowthrough by ELISA).

    The Rat-Fc is present because I can detect the protein by WB, ELISA and FACS using anti-Rat secondary Ab's. Also the size estimation by WB implies that the Rat Fc is not missing (although the protein runs 2-3 KDa higher than I expect).

    I also tried purification by protein A/G agarose beads and a different column to exclude problems with the particular column I used. I use a phosphate buffer (pH 7.0) as a binding buffer and I directly apply filtered cell culture medium (adjusted to pH 7.0) to the column.

    Basically my question is whether I should be worried about the funtionality of the Fc and how it can be that the protein is not bound by protein G.

    Thanks in advance,

    Niels Dammes

    Niels Dammes · Tel Aviv University

    Thanks for your reply. I transfect in serum free medium so I think that the albumin is not a problem. I produce the protein in Expi293F which are optimized for a specific serum free medium.

    When I do my analysis I test both culture medium and cell extracts. It is secreted even though there is also a lare amount of protein still in the cells. But the amounts are anyway very high so there is more than enough protein in the medium.

  • Heide Marie Resch added an answer:
    Is it correct to consider a protein nuclear extract more crowded than a solution of a recombinant protein?

    Hi to all,

    I'm trying to study a protein DNA complex.

    when I try EMSA i have positive results with the nuclear extracts, while with the recombinant protein I have never seen any interaction.

    On the contrary when pull down assays with streptavidin magnetic beads are performed, I have good result both with recombinant and endogenous protein.

    I know for sure that the protein that binds the dna probe within the nuclear extracts is not post translationally modified, because I performed a previous trypsin Digestion and a Database search for PTMs.

    So i'm wandering if the positive results with the recombinant protein and pull down are due to a sort of crowding effect given by the beads, that simulate the environment of the protein in nuclear extracts.


    Heide Marie Resch · NanoTemper Technologies GmbH

    You could easily quantify the binding affinities with recombinant protein and nuclear extracts by using MST (MicroScale Thermophoresis), in free solution, without any surface immobilization. In the review attached, the binding affinities for a protein-protein interaction with purified proteins and in cell lysate were basically identical, however, we also see differences if other components present in the cell lysate interact with one of the binding partners. Comparing binding affinities in buffer and in cell lysate thus provides interesting insights into biomolecular interactions.

  • Csaba Zoltan Kibedi Szabo added an answer:
    How can I purify protein with 27 KDa from GST ?

    Dear colleagues, i am trying to purify a protein with 26 kDa size that is expressed as a GST fusion protein. When i have done  on column GST cleavage in the flow through both GST and my protein of interest coming together. Even i tried Superdex 200 SEC there also proteins are co-eluting. My protein pI is 4.54 and GST pI is 6.09 so i did ion exchange (mono q) with buffer pH 6.5 i eluted by gradient elution with 1M NaCl but here also both the protein coming together. Please suggest some idea to purify the protein. I need this protein for crystallization and structural studies.

    the image i have given is MONOQ profile in 18% SDS gel 

    Csaba Zoltan Kibedi Szabo · Max Planck Institute for Biophysical Chemistry

    Dear Rajivghandi,

    Are you sure that the lower band is GST contamination? I mean did you do mass spec? Actually for me for me it seems rather a degradation..if you carefully check the first gel which you uploaded the contaminant seems to migrate higher when compared to GST (see fraction 5). Also notice that in lane 6 you actually have two bands (contaminants). I would suggest to destain better your gels in order to see clearer what you have there. On the other hand in the second gel you have a number of contaminants which also indicates that your protein is prone to degradation.

    Regarding the purification on MonoQ, it is hard to believe that you can't separate your protein from GST. MonoQ column is very high resolution column therefore if you do a proper gradient, for sure you will be able to separate it. As a control you can add back GST to your protein and then load on MonoQ and perform a proper gradient in order to get two defined peaks and then check your protein. If you still have this lower band that's a good indication that is a degradation.

    The S200 column was not a really good idea as it doesn't have the necessary resolution required in your case, not even the S75 in my opinion, therefore the only option is to play with the MonoQ (i.e the gradient).

    Before going for recloning I would suggest to do limited proteolysis, perhaps this way you will manage to identify a more stable fragment resulting better crystals.

    Good luck!  

  • Rhudith Cabulong added an answer:
    What could be the reason for no Protein Expression after cloning?

    I inserted my gene in pACYCduet-1 MCS2, sequenced the construct and the sequence was fine. Everything was OK. However, when I tested its expression by running SDS-PAGE, the result was negative. I induced my culture with 0.5mM IPTG after 0.3 OD600. What could be the problem?

    Rhudith Cabulong · Myongji University

    Thank you everyone for your suggestions! I already found out the cause of the problem. Yes Christian, it is my expression host that has a problem. lambda DE3 gene was not integrated into the chromosome of my host.

    Thank you everyone for the suggestions.

  • Veronika Chromikova added an answer:
    Can the IgM J chain on SDS-PAGE be detected?

    While characterising purified antibody on SDS PAGE I ended up with only one band of approximately 14kDa, similar to the J chain, though no heavy or light chains. Does anyone know if you have the J chain present without the main heavy and light ones? More specifically if the purification method used was actually meant to bind to the light chain?

    Veronika Chromikova · University of Natural Resources and Life Science Vienna

    I think you are not providing enough information to answer your questions. It would be useful to know, what is the production system you use to produce your IgMs and what is the method used for purification?

    It is possible to detect J chains on SDS- PAGE, but if what you loaded were indeed IgMs, you should be able to see also bands for HC and LC (and those should be much thicker than the band for JC). I would absolutely go for Western blot though, because with the size of ~14 kDa, it is well possible that you only have some kind of contamination not an IgM. It sounds to me as if the purification failed and you didn't elute IgM at all.

    I assume you are producing secreted pentameric IgMs? Then alternatively, you could try non-reducing gradient 4-12% PAGE gel to run your samples. There you would be able to see pentamers/hexamers and all the other smaller fractions and you would see pretty clearly if what you purified are IgMs.

  • Van Anthony M Villar added an answer:
    What can be the reason for the trouble with Dopamine receptor extraction and subsequent western blot experiments?

    I've been using the "Mem-PER™ Plus Membrane Protein Extraction Kit" (Lifetechnologies) to isolate the membrane-associated protein fraction and also all integral proteins from mouse tissue according to the manufacturers protocol,
    After loading a certain amount of protein (with an loading dye containing SDS in a concentration of 5% related to the whole sample and DTT ) on the SDS PAGE, i was not able to seperate the proteins by electrophoresis (almost all proteins remain in the upper third section whereas the marker migrate properly).

    I used a stacking gel (4% polycacrylamide ; pH 6,8) and seperating gel (7% ,10% respectively ;  pH 8,8) with following settings:
    100V for 20 min (stacking)
    180V, 130V respectively for a certain period of time (separating)

    Has anyone an idea what i can do to solve this problem? Or what can be the reason for this poor-migration behaviour?
    Normally dopamine receptor can be detected at 40-60 kDa, my results shown a band at around 180-250 kDa, indicating  that proteins were not (badly) seperated.

    Thx a lot for your help

    Van Anthony M Villar · University of Maryland, Baltimore

    We work mainly on dopamine receptors and we never have problems with IB. As previously noted, the extraction kit you were using is gentle enough to preserve the protein complexes, which may be beneficial for protein-protein interaction assays. A regular RIPA buffer will disaggregate the complexes, otherwise, supplement with urea or more SDS or b-mercaptoethanol.  You may also want to use a membrane-impermeant biotin to extract proteins on the surface of plasma membranes. Lastly, do the Abs you're using work? Have you validated their specificity?

  • Michael Urban added an answer:
    Can someone suggest a detergent for solubilizing a membrane protein effectively?

    I have used DDM, Cant use SDS

    Michael Urban · Goethe-Universität Frankfurt am Main

    with regard to what M Joanne Lemieux wrote above:

    how were your results using DDM? Is the "trouble" you have (I guess?) with the detergent, or protein purification per se in your case? If you could give additional information the people here are surely glad to assist :)

    Factors like cell disruption, membrane isolation, protein purification, buffer and storage conditions can greatly influence your successful solubilization and need to be checked for every individual protein (sadly)

  • Adam B Shapiro added an answer:
    Any suggestions about concentrating a protein solution in a centrivap?

    I recently performed an ELISA for TNF-alpha using undiluted cell supernatant and my protein concentration was too dilute to get decent results.  Would using a centrivap to concentrate my protein followed by reconstitution in a smaller amount of solvent work help?

    Adam B Shapiro · AstraZeneca

    My concern is that the high ionic strength will reduce the binding affinity of the capture antibody. The salt will be washed away after that step. Do you know what the salt concentration is in the medium, and by how much you will concentrate it?

  • Adam B Shapiro added an answer:
    How do I extract protein from a matrix containing polymers, which has the similar molecular weight with protein?

    I am trying to separate the protein from a mixture containing HPMC. The solution is also viscous.

    Adam B Shapiro · AstraZeneca

    Dilution of the protein sample should not be a problem in the method I suggested because the protein can be eluted in a concentrated form with a high-salt wash. To simplify sample processing, you could use pre-made ion-exchange resin-filled 96-well plates such as these (, or make something similar yourself with bulk resin.

  • Ke-Wei Zhao added an answer:
    Does anyone have experience on Yap1 detection on western blot?

    I'm trying to detect Yap1 protein both on mammalian cells lysates as well as a recombinant protein. The band I can visualize runs approximatively at 35-40 kDa on a 12% SDS-Page gel, under reducing conditions, while I would expect it 60-70 kDa. The issue is with two different antibodies from different companies. Last detail: if I search for the phosphorylated variant, the signal is approximatively at 60 kDa, the right molecular weight.

    Ke-Wei Zhao · U.S. Department of Veterans Affairs

    I use Pierce's EZ-link activated peroxidase (#31487).  It can be part of a kit as well.

  • Jürgen Denecke added an answer:
    Is there any protocol for secretory protein enrichment from fungal culture (Ascochyta rabiei), to be used in western blot experiments?

    These secretory protein would be used for relative expression studies.

    Jürgen Denecke · University of Leeds

    You can use spin dialysis (for instance centricon filters) to concentrate the proteins in the medium without having to precipitate (and loose activity). Essentially, you pipet the diluted culture medium into the filter, centrifuge it, and water + solutes below a certain molecular weight (i.e. 10 kDa) pass through the filter, larger proteins stay in the medium on top of the filter in a more concentrated form. Use a refrigerated centrifuge to avoid proteolysis, fungi secrete loads of hydrolases. You can easily concentrate 2 ml of medium to just 200 microlitres in half an hour, that is a 10-fold concentration. The filters come in different sizes and different molecular weight cut-offs (3 kDa, 10 kDa, 30 kDa...)

  • Kurt D. Berndt added an answer:
    Is ammonium sulphate or acetone precipitation the better method to isolate and concentrate proteins?

    Kindly, brief me about the acetone ppt protocol. 

    Kurt D. Berndt · Karolinska Institutet

    Hi Sumit
    The (NH4)2SO4 will cause artefacts in SDS PAGE. We are back again to why you want to precipitate. If it is purely part of an analytical procedure, then acetone precipitation is a good way to go. It scales down very nicely (Analytical Biochemistry (1984) 138, pp 141–143) so small amounts can be recovered in microfuge tube size samples. Dried over a stream of N2, there is no residue and samples are fit for SDS PAGE:

  • Edward Michelini added an answer:
    How can I remove DNA from nuclear pellet with polyethyleneimine for protein analysis?

    The nuclear pellet was obtained through low speed centrifugation after cell disruption and resuspended in lysis buffer. How to get rid of the remaining DNA to conduct a western blot with the lysate?

    Westerns work just fine with DNA present, are you needing DNA absent for another reason?

  • Bhumika Bhatt added an answer:
    Lipidation of proteins on SDS-Page

    Hi Everyone,

    I would like to know how a lipidated protein runs on a SDS-PAGE gel. Is it true that it is cleaved on lipidation site? If yes, is there a way to stop the cleavage and also if this cleavage happens in the cells in physiological condition. If not, how does a lipidated protein size vary on a gel?

    thank you


    Bhumika Bhatt · Victoria University of Wellington

    Well if you're doing a 1D gel pI is irrelevant but if you're doing 2D, just use a narrow range 6-11 strip and you should be fine.

  • Sugata Roychowdhury added an answer:
    How can I separate monomeric protein from its oligomer when both comes out in the flow through?

    I am working with HIV-1 gp120 Env protein gp120. Its theoretical pI is 7.91 although it is heavily glycosylated and is being expressed in tobacco plants. I am purifying it sequentially through TALON IMAC, Galanthus nivalis lectin resin, DEAE anion exchanger. After DEAE run, I see both monomer and oligomer (formed probably due to inter-molecular disulphide bridges as it is reduced in reducing gel) in Flow through. How do I separate the two bands? My thoughts are to use Size-exclusion Chromatography to separate monomeric (Mol. wt 120 KDa) from the oligomeric (~ 250 KDa) as seen in non-reducing gel. Does anyone has any other thoughts on this? Please see attached slide of the gel picture.

    Sugata Roychowdhury · Indiana University Bloomington

    Thanks all for the comments. At this point, I am not sure at what stage the oligomers are formed and that using DTT might break the intramolecular disulphide bonds present in the monomer, thus making it non-functional. But definitely, I would like to use Superdex 200 to see if I could separate the two. For Deae resin, I have been using 20mM phosphate at pH 8. Thanks Kristin and Chris for the documents. Also, I am planning to do DLS on the FT to see what percent of oligomers I have in the mixed sample.

  • Timothy A Reinhardt added an answer:
    Is there any possibility to resolve the presynaptic membrane proteins in its native condition?

    I'm successful in separating the presynaptic and PSD fraction using a method that published in a Neuron article. I have some native gel running complication with the pre-synaptic membrane fraction as the gel goes bizarre. I found that the triton X-100 in the presynaptic fraction causing this while running native page gel. I even found the same while running in SDS/PAGE but I was successful after subjecting the presynaptic fraction to acetone precipitation.  But the acetone precipitated presynaptic fractions is not compatible to resolve you native proteome as acetone disturbs the lipid bilayer thus no longer the native condition of your membrane proteins maintained. At the separation step of presynaptic and PSD fraction we use 1% triton at pH 8. During this step the presynaptic fraction is resolved in the supernatant along with 1% triton. I used to filter (used 100 K esp for running native page also used 10 K to 30 K filters) and exchange the presynaptic fraction buffer composition (ph 8 to 7.4) to get rid of triton but instead it gets concentrated to form a micelle I persume so. How do I get void this triton from this fraction without affecting the nativity of a proteome? Does anyone have any idea how do I over this situation?  

    Timothy A Reinhardt · United States Department of Agriculture

    Consider Blue Native Gel Electrophoresis which separates membrane protein complexes in their native form.

  • Soha Telfah 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?

    Soha Telfah · Philadelphia University Jordan

    thanks ahmad, i dont think i can use TFA or acetic acid, instead phosphate buffer pH=3 is commonly recommended, and for isopropanol the company never recommend using it, maybe methanol or acetonitril gradient. 

  • Tobias Zuest added an answer:
    What is the best 96 well plate sealing film suitable for HPLC?

    I have until now been been slightly unruly using adhesive foil plates to seal 96 well sample plates ready for loading onto our HPLC (Alliance 2795). Whilst this is adequate for my needs and I don't mind cleaning the injection port once in a while I'm probably getting leeching of materials from the small amounts of adhesive which find their way into the injection port or perhaps even the injection loop. I'm monitoring my samples via MRM so this slight contamination isn't a problem but I would rather have a method which keeps my system as clean as possible. Does anyone have any recommendations for films that seal 96 well plates that are neither foil nor use conventional adhesive and aren't strong enough to damage the needle or mechanism? Thank you.

    Tobias Zuest · Cornell University

    Hi Marcus,

    My samples are in 100% MeOH, and most sealing films that I've tried would come off within a couple of minutes. I've also tried the regular adhesive foils that seem to be working for you, and those came off within a couple of hours. So maybe the difference in solvent is enough that you won't have that problem.

    Good luck,


  • Gunjan Dhawan 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

    Gunjan Dhawan · University of North Dakota

    I have seen my peptide aggregate in HFIP also. So be careful when using it.

  • Wojciech Wozny added an answer:
    Why does my picking 2D gel not look similar to my analytical gels?

    50 µg of whey proteins were labeled with Cydyes on 3 replicate analytical gels and I did the Decyder analysis for them, however when I loaded 400 µg on preparative gel for picking, my spots of interest don't look the same.

    Wojciech Wozny · Wrocław Research Centre EIT+


    I agree with Stephen, we do the same. We use not  DIGE  but Proteotope  ( Instead,   CyDyes  we use iodine isotopes following differential radioimaging.  For preparative gel,  we spike  radioactive  proteins with preparative  amounts of the protein extract.


  • Janet Lee Smith added an answer:
    How do we equilibrate the millipore protein concentrator while using it for the first time?
    Can it be reused, and if yes, how many times?
    Janet Lee Smith · EMD Millipore

    @Amritlal Mandal:  For clarification, the EMD Millipore YM membranes do NOT contain sodium azide as purchased.  Prerinsing is only necessary to remove glycerol humectant if this interferes with downstream applications.

  • Vanitha Selvarajan added an answer:
    Is it possible to assess the extent of phagocytosis in a macrophage-tumour cell co-cuture? If so, what methods can be used?

    I am doing an experiment where I use a recombinant protein to activate and proliferate macrophage so as to reduce the tumour cell mass through a co-culture based model. In this case, I need to assess the extent of killing of tumor cells by macrophages.

    Vanitha Selvarajan · Indian Institute of Technology Guwahati

    Thank u so much for your replies. I'll try to carry out these procedures.

  • Spencer Berg added an answer:
    How can we lyse C6 cells which are glial cell lines of rat origin (cancerous cells)?

    I am trying to lyse C6 cell lines which are cancerous glial cell lines of rat origin. Kindly let me the best way to achieve cytosolic and nuclear protein?

    Spencer Berg · University of Waterloo

    I take it you're trying to collect nuclear and cytosolic protein fractions for western blot or some other downstream application? I use a nuclear lysis kit from Millipore (product number 2900) that does the job quite easily. There are probably many other commercial alternatives, or homemade buffer recipes that will work.

    Basically, after collecting the cells you lyse them but leave the nuclei intact. Spin down the nuclei and collect the supernatant (your cytosolic fraction). Resuspend the nuclei in another buffer (nuclear lysis buffer), lyse them, spin down the membranes, and collect the supernatant as your nuclear fraction.

    For the actual lysis, the kit recommends passing the suspensions through a syringe, but we've also successfully used a few pulses with a probe sonicator. 

    Good luck!

  • Edward Michelini added an answer:
    What should I do with this issue in phase separation during the concentration of a protein?

    I'm trying to use a 12kDa protein to set up crystallization. While I was using the ultra-centrifuge tube to concentrate this protein, I noticed some phase separation, and the concentration of this protein can not be increased anymore.

    The Grand average of hydropathicity (GRAVY) of this protein is -1.553. The buffer I used contains 20mM Hepes, pH 8.2, 50 mM NaCl.

    I'm wondering whether the salt concentration I used is too low. 

    Did anyone have this problem with me? Any suggestions? Thanks

    Perhaps you are referring to the concentrated protein "gel layer" which I see often when I am using dead end MWCO spin filters? This (sometimes colored) layer is set up right at the membrane surface but does not represent an insoluble aggregate. I simply mix the upper buffer chamber well with a pipette and proceed with the concentration spins. The schlieren lines are visible until the solution is homogeneous, at which point the filter again passes permeable solute.  Vortexing can also work if the upper buffer is secure. I see this as a sort of intermittent tangential flow required for efficient filtration.

  • Chao Hsu-Wen added an answer:
    From the attached 2D gel picture, can you tell me where I made a mistake?

    Hi everyone

    From the attached 2D gel picture, where I did a mistake?  How can I improve my result?

    I have extracted total protein from rice leaves with TCA acetone method, I want to check the differential expression of proteins in rice leaves after treated with protein eliciter from fungus (M. Oryzea

    I have used the IPG strips (PH- 3 to 10) and 12.5 % SDS PAGE  

    Chao Hsu-Wen · Academia Sinica


    I agree with Massood, you need to remove ions as possible as you can from your sample before IEF. And it is better to decrease amount of protein for IEF. Moreover, the well you make for marker loading is not good, you need to prevent the leakage of marker to other region. And since you use silver staining for protein detecting, you do not need to load so huge amount marker. It is better to decrease to 20% to 30% from your original amount.

  • M. Farooq Wahab added an answer:
    How do I determine t0 (void volume) of a HILIC column?

    Since the mechanism of HILIC is not fully understood, I was wondering how to determine the dead time/void volume/t0 of a HILIC column (in particular amide, zwitterionic)?

    M. Farooq Wahab · University of Texas at Arlington

    The dead time marker in HILIC or in any other mode of chromatography is certainly a non-trivial problem, since the knowledge of stationary phase chemistry is quite critical. Polymeric phases (e.g. ZIC HILIC, Poly LC phases) will swell and contract with respect to water content and the dead time will change. Knut Irgum has a generic way of determining t0 in HILIC mode. Please see J. Chromatogr. A, 2013, 1320, 33-47, you will see that there is probably no universal way of determining t0 for all HILIC columns.

  • Daniela S Goncalves added an answer:
    Is there any protocol to remove virus from insect cells?

    I am working with Lutozmyia longipalpis cell line - Lulo, and seems these cells are infected with some virus (I don't know exactly what kind of virus it is/ there are). Due to this fact, I would like to know if there is any protocol to remove virus from insect cells.

    Daniela S Goncalves · Fundação Oswaldo Cruz

    Hi Archit!

    I want to use this cell without any virus because I'm trying to infect them with another intracellular pathogen and maybe this virus is blocking the infection.

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