A forum to address questions regarding methods of protein purification.

• Tomáš Hluska

  Is anybody here able to identify a protein from gel in a week?

  I have purified an enzyme and I need to identify it. People at our department are not able to do it always for some reason. Recently they've got some peaks, but were not able to identify the protein.I have purified an enzyme and I need to identify it. People at our department are not able to do it always for some reason. Recently they've got some peaks, but were not able to identify the protein. So, would there be anyone able to take the care and identify my maize protein from gel in let's say one week?

  

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    Sothinathan Vijay Anandavijayan replied

    If you are a member of Linkedin then you can post your question on the discussion groups like purification-biologics, I am sure you will receive more response. Mass spec will be the quickest way ofIf you are a member of Linkedin then you can post your question on the discussion groups like purification-biologics, I am sure you will receive more response. Mass spec will be the quickest way of identifying proteins/enzymes etc.

    2 days ago
• Sugumar Paramasivam

  How to check protein purity without using gel electrophoresis?

  Without gel electrophoresis method, how to calculate protein purity.
• Lauren S Sherman

  How can I desalt a protein sample currently in >300mM salt?

  I have a protein family with a histidine rich region that I am successfully isolating by metal affinity chromatography. I have previously run these samples on one-dimensional SDS-PAGE gels followedI have a protein family with a histidine rich region that I am successfully isolating by metal affinity chromatography. I have previously run these samples on one-dimensional SDS-PAGE gels followed by Western blot, and am now looking to run the samples on two-dimensional gels. This requires that I remove the majority, if not all, of the salt. My original sample is in 400mM salt (in theory, I think this salt content should get washed out during the metal affinity isolation process), and the final buffer from my metal affinity isolation uses 300mM salt (in 10-20ml total volume). I have tried using Pierce concentrating/desalting/buffer exchange columns (http://www.piercenet.com/browse.cfm?fldID=E32DC01E-F86C-3C33-6099-439DE8CAAC85 ), but that does not seem to remove enough salt. I'm considering running the concentrated sample through Bio-Rad Bio-Spin p6 desalting columns as a second desalting step (http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=BRCatgProductDetail&productID=211001 ). (I've also tried dialyzing my sample into deionized water, but I had high protein loss.) Does anyone have a suggestion for efficiently getting rid of this much salt? My proteins range from 40kD-250kD in size. I am primarily using gravity filtration, but I do also have a simple pump. Thanks!

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    David J Lee replied

    If your protein is easy to purify - which it sounds like it is due to its inherent NiAg binding properties, why don't you next time just make your imidazole elution buffer with low salt. Or do aIf your protein is easy to purify - which it sounds like it is due to its inherent NiAg binding properties, why don't you next time just make your imidazole elution buffer with low salt. Or do a step-wise wash with decreasing salt concentrations - then elute in low salt

    4 days ago
• Amaury Pupo

  Immunoprecipitation: Immobilized antibodies, magnetic pearls, free antibodies and protein A/G columns. Which is your choice?

  I would like to know about practical experiences in protein immunoprecipitation. I am interested in the immunoprecipitation of MHC/peptide complexes from eukaryote membranes, but any suggestion orI would like to know about practical experiences in protein immunoprecipitation. I am interested in the immunoprecipitation of MHC/peptide complexes from eukaryote membranes, but any suggestion or comment for any kind of protein will be welcome.

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    Ryan Huyck replied

    Any of those options are good, it just depends what you have available in lab. The start up costs and equipment for a magnetic bead system is a bit more than the others, but it's also the easiest,Any of those options are good, it just depends what you have available in lab. The start up costs and equipment for a magnetic bead system is a bit more than the others, but it's also the easiest, since it keeps the beads from being lost as easily during washes and such, and is very rapid. You can also use a spin column like system from Thermo Pierce. You can either use Protein A/G pre-linked beads, or have a handy easy procedure to covalently couple whatever you want to them. Washing and everything is easy, since liquid flows through the column, and the resins can be reused many times; no worry about accidentally sucking up beads or not getting all the liquid out. Since you're working with membrane proteins, I would recommend not trying to directly couple them to the resin as there's a chance you'll block the solvent exposed active site, and instead use antibody or antibody-protein A/G linked beads.

    5 days ago
• Kalpana Hiteshi

  How to purify a cold active alpha-amylase from psychrophilic isolate?

  I am working on psychrophiles and I have to produce cold active alpha-amylase from that. The incubation time for maximum production is about 20-25 days from a few isolates I have got to date. This isI am working on psychrophiles and I have to produce cold active alpha-amylase from that. The incubation time for maximum production is about 20-25 days from a few isolates I have got to date. This is taking a very long time. If anybody have some suggestions for this work, kindly let me know. I also want to know what can be the possible applications of this enzyme, which are possible to perform at lab scale.

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    Dhananjay Singh Mankotia replied

    What is the purpose of producing such cold active alpha-amylase? Are they useful to any Industry? Research? As for your problem, I understand psychrophiles are an obvious choice for isolating such aWhat is the purpose of producing such cold active alpha-amylase? Are they useful to any Industry? Research? As for your problem, I understand psychrophiles are an obvious choice for isolating such a cold tolerant enzyme but production of enzymes in psychrophiles in inherently low and its isolation/purification can be really tricky. So you will face problem.

    7 days ago
• Nieves Aranda

  Protein purification by immunoprecipitation from Arabidopsis

  I'd like purify proteins by immunoprecipitation, from Arabidopsis mutants overexpressing an HA-tagged protein. For this purpose I'm doing total protein extraction and then purification byI'd like purify proteins by immunoprecipitation, from Arabidopsis mutants overexpressing an HA-tagged protein. For this purpose I'm doing total protein extraction and then purification by immunoprecipitation. Has anyone had experience with immunoprecipitation? Which is the best way, using anti-HA affinity agarose, or using protein A/protein G Agarose? Which kit do you use?
• Prem Prakash

  Can anyone explain how I will know that my protein is in hexameric form?

  And if mutation has been done (in protein gene) and then there is no activity found with enzyme how one can monitor the function after mutation. Please explain both question if its possible.

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    Prem Prakash replied

    Thank you Jens....:)

    12 days ago
• Alessandra Morana

  I want to precipitate proteins from a culture medium of a halophilic bacterium with ammonium sulphate. Does someone know if NaCl present in the medium can interfere with protein precipitation?

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• he Wanglin

  Methods to extract the protein of interest

  An engineered bacteria was built by formers. I just did the expression by IPTG, and want to extract the interest protein for further study. The only thing I know about the interest protein is that itAn engineered bacteria was built by formers. I just did the expression by IPTG, and want to extract the interest protein for further study. The only thing I know about the interest protein is that it is about 108 kda.What would be the best method for extraction?

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    Enrico Ferrari replied

    I would be really surprised if they cloned a recombinant protein in E.coli without an affinity tag. Try to figure out which expression vector they used and then select your resin for affinityI would be really surprised if they cloned a recombinant protein in E.coli without an affinity tag. Try to figure out which expression vector they used and then select your resin for affinity purification accordingly. The tag can be his-tag, GST, MBP or whatever, depending on the expression system they used. Hopefully you don't even need recloning.

    Apr 25, 2012
• mj mm

  Protein tags

  I wish to do a pull-down assay of a protein (chromatin modifier) which is 520 kda. It is cleaved into 180 kda and 320kda units. Can anyone suggest a suitable tag system for the protein? What are theI wish to do a pull-down assay of a protein (chromatin modifier) which is 520 kda. It is cleaved into 180 kda and 320kda units. Can anyone suggest a suitable tag system for the protein? What are the considerations in choosing a tag for a protein for such experiments?

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• Czuee Morey

  How to purify a protein with pI in the physiological range?

  Hi, I am trying to purify a protein whose pI is in the physiological range, which is generally the pH range for most buffers and growth media used in the lab. Does anyone have any tips on how IHi, I am trying to purify a protein whose pI is in the physiological range, which is generally the pH range for most buffers and growth media used in the lab. Does anyone have any tips on how I could purify this protein. I actually tried to express and purify this protein, but ended up isolating a bacterial protein artifact! From the results of my first analytical purification, it seems that because the protein is expressed in LB (which I guess has a pH near 7) it aggregates soon after expression. I tried changing the pH of the lysis buffer (between 6.0 - 7.4) but the protein is still not soluble! I guess the protein is getting aggregated either due to misfolding (it has 16 Cys residues!) or because the pH of the growth medium isn't suitable. Should I change the media or adjust the pH of LB with buffer? What pH should be used so that the bacteria can grow happily and my protein doesn't aggregate? The expression and purification buffers I used earlier were as follows: Growth medium : LB Induction : 1mM IPTG Lysis buffer: 20mM Tris-HCl pH 8.0, 100mM NaCl, 1mM DTT, 1mM EDTA, MgCl2, PMSF, lysozyme, TritonX, DNase I tried changing pH of lysis buffer but this did not help Any help, suggestions and comments are welcome!

  Recent replies ⋅ Show All (18)

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    Tomáš Hluska replied

    Is all your protein eluted with 8 mM imidazole? In such case, you can elute it with only say 10 mM imidazole and the rest of bound proteins elute independently in other fraction.

    Apr 11, 2012
• Sheraz Bhat

  In the process of purification we use ammonium sulphate fractionation and then subject the samples to dialysis to remove the salt and carry on further processing like gel filtration, affinity etc. toIn the process of purification we use ammonium sulphate fractionation and then subject the samples to dialysis to remove the salt and carry on further processing like gel filtration, affinity etc. to achieve several fold purification and then PAGE. If the said salt is present it gives diffused bands rather than the desired sharp ones. In this regard I want know how can complete dialysis be checked, and also if there is any better method not involving this ammonium sulphate fractionation.

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    Tomáš Hluska replied

    ionex is not very suitable if Sheraz wants to desalt the sample. It would be better to go directly to the gel filtration chromatography.

    Apr 9, 2012
• Loreto Carvallo

  Co-IP using crosslinking

  I am trying to do a Co-IP, I think that my interactions is very weak and/or transient, because I can't see anything by regular Co-IP. Anyone use or know a protocol for Co-IP using crosslinking?

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    Loreto Carvallo replied

    Thanks a lot manjapra!!!!

    Apr 4, 2012
• Kritika Sudan

  Insoluble protein aggregates during elution under native conditions

  I was in the process of islolating a recombinant protein under native conditions from E.coli . During elution of the protein, an insolube aggregate of proteins was seen with the help of using anI was in the process of islolating a recombinant protein under native conditions from E.coli . During elution of the protein, an insolube aggregate of proteins was seen with the help of using an elution buffer which contains 300mM NACL, 250mM Imidazole and and 50mM NAH2PO4. I would be grateful if anyone could provide some advice or suggestion for dealing with this problem of protein aggregates.

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    Paola Di Bonito replied

    Thank you Kritika now your experience is useful to us all

    Mar 25, 2012
• Rekha jagdish Tripathi

  Purification of protein from grapes?

  I am using trichloroacetic acid-acetone precipitation and phenol precipitation method for protein purification for 2D -pH 3-10..strip were used ,but I am not getting any dot on gel? Any suggestions?