Questions tagged [purification]

The molecular technique of isolating specific molecules, especially biomacromolecules, from complex solutions.

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qPCR precipitation

I have performed DNA precipitation using two methods on an amplified 194 bp PCR product: using a PEG method with just cold 80% ethanol PEG worked much better for me. Can the same methods be applied ...
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Can I column purify instead of gel extract?

I'm digesting a plasmid for assembly. In my experience, I always have low yields when gel extracting and purifying. Since I am only making one cut in my vector, could I just purify with a column? Any ...
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Mass Spectrometry Artefacts: R-deamidation / Oxidation?

Context: I am a student-researcher. I expressed GST-tagged protein (Pyruvate Kinase) in the yeast Saccharomyces cerevisiae on synthetic drop-out media. I then purified the GST-tagged protein, ran it ...
thefutureisnow's user avatar
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Heat-treatment for protein expression

I want to express thermophilic protein in E. coli and the first step purification is to heat the crude protein. Since I don't know the optimum temperature to denature the contaminants, I plan to heat ...
anastaciaa's user avatar
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What's the best way to purify my His tagged protein? Supernatant super viscous after first sonication?

I am trying to purify my his-tagged protein of interest, disulfide isomerase. It is about 40kDa and is cloned in pET28a vector, at XholI and NdelI, and expressed in BL21. I'm having issues with my ...
Yoshokie's user avatar
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Why are IMAC and gel filtration combined?

In a practical course I used an Biorad Profina system to purify a protein with a histidine tag. The device uses a column for IMAC and one for gel filtration. Why are these two devices combined in one? ...
Mourinho_1's user avatar
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Which is more efficient as a germicide: UVC, Ozone or combination and why?

I've read many contradictory info about ozone vs UVC light irradiation and cannot come to a conclusion, I am totally confused. As it commonly known UV germicidal irradiation is very effective against ...
Suncatcher's user avatar
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Appropriate regeneration of StrepTrap HP columns for FPLC

My question is related to protein purification using a ÄKTA FPLC. We used StrepTrap HP Columns (1 ml column Volume (CV)) from GE Healthcare Life Sciences to purify a strep-tagged protein. In the first ...
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Fluorescence assays to identify protein concentration without adding a large peptide sequence?

I'm trying to find a way of tagging a protein with something visually quantifiable to track protein concentration through potential purification steps and screen for the most efficient such steps. ...
Tal's user avatar
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Separate DNA fragments with very different size (oligo and lambda-DNA)

I am running a ligation reaction to one of the sticky ends of lambda-DNA. The oligo for the ligation is 25 bp (10x excess), whereas lambda-DNA is 48 kbp. I would like to recover the lambda-DNA and ...
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What buffer should I choose for IEX chromatography for purifying IgY

I will use ion exchange chromatography with an anion exchange column to purify chicken IgY. Prior to this I did dialysis to remove salts from previous purification steps, is it possible after this ...
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Why does 4-thiouracil labelling to map RNA-binding proteins cause a T-C change?

I am now reading a paper about the purification and identification of mRNAs and its RNA binding proteins by using UV crosslinking and immunoprecipitation. I came upon this sentence which puzzled me ...
Michaela14's user avatar
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Ligation without purifying insert

I am planning to insert a 45 bp sequence in a vector. After restricting my insert to create compatible sticky ends, I am finding no way to clean it up. Is there any way for cleaning this fragment ...
RKK's user avatar
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Centrifuge after sonication

I follow a protocol to get protein from E.coli cells after sonication. I used to grow 6 litres of large cultures and add IPTG to express the protein. I centrifuge for 10 mins at 8,000 rpm and get the ...
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False positives in TAP - MS experiments

Is anyone aware of a website where they show common false positives often found when doing a TAP-MS experiment to find protein-protein interaction experiments? Particularly the Acs1 protein (Acetyl-...
Loko's user avatar
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What is the protocol for extracting Protease Onion?

I am Investigating the protease concentration in certain fruits and vegetables. I am unable to find the protocols for the extraction from onions. I want to purify my protein using the ammonium ...
TescoExpress's user avatar
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Laemmli-SDS-PAGE problems [closed]

I did Laemmli-SDS-PAGE for my Ammonium sulphate precipitate but I had very weak band and have very weird part at the end of gel. Please help me to solve that problem. Thanks
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How do I look for pathogen in water with a microscope? Is it possible?

I am a 9th grade student from Manila and we're currently working on an investigatory project. We want to know how effective SODIS or solar water disinfection is. The purification method claims to be ...
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Dyes that won’t bind to dna

I am looking for a dye that won’t bind to DNA. I want to add dye to proteinase K to make is visible when adding to clear buffer liquid samples. I need it to not bind to the DNA so it will be removed ...
Lindsay's user avatar
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Methods for phage separation

How can a mixture of unspecified phages be separated (into singular phage strains)? I.e, what are the main methods? My research shows it can be done using CsCl centrifugation and affinity ...
Zubo's user avatar
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Ion-exchange vs Gel Permeation Chromatography for proteins

For extracting Lysozyme from egg white which method would you recommend and please help me understand why. Lysozyme has a higher isoelectric point (pH 10.5) and a lower molecular mass (14 307 Da) ...
Cello_101's user avatar
3 votes
2 answers
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Dissolving cell pellet after sonication

I was doing a protein prep and I made a mistake. After sonicating my cells, I was supposed to centrifuge and collect the supernatant (my protein is soluble and comes in the supernatant). I however, ...
Chem-man17's user avatar
3 votes
1 answer
200 views

Sephadex column for alpha-amylase

I want to purify crude alpha-amylase with column chromatography. I am using a spehadex 75, But for some reason I can't find any information on how to make the slurry. I can quickly find tons of ...
LazorLord's user avatar
3 votes
1 answer
133 views

Can one use dialysis tubing several times?

I would like to know, if one can use dialysis tubes multiple times. Or is there a risk of plugging the pores? I use the tubes for dialysis of a solution that contains a precipitated enzyme (183 kDa) ...
Luke's user avatar
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How to dialyze large amounts of precipitated enzyme solution (ammonium sulfate)?

I am no biochemist, but I want to purify some liters of solution through dialysis. What I know are those dialysis tubes in glas vessels on stir plates for labs. But how is this done in industry for ...
Luke's user avatar
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Measuring protein concentration, Bradford vs. Nanodrop?

I know that the bradford assay is a very standard way of measuring protein concentration after e.g. a purification. However, in the lab that I work in now they normally only use nano drop at the 280nm ...
CuriousTree's user avatar
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1 answer
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Protein purification: Linear elutions or stepwise elution, using imidazole or pH gradient?

I now work in a lab with a protein and a pre-made purification protocol, were they purify a His-tagged protein using linear elution which then gives a steady increase in the imidazole concentration. ...
CuriousTree's user avatar
3 votes
1 answer
6k views

Clarify pre-column pressure vs. system pressure vs. backpressure for prepacked columns used with the äkta purification system

Background: I am using the Äkta pure system for protein purification, with prepacked columns from GE-healthcare. Just to take one example, I use the HisTrap 5 mL column (IMAC: immobilized metal ...
CuriousTree's user avatar
1 vote
1 answer
5k views

Help analyzing SDS-Page gel

In this experiment, we transformed a truncation of the NFAT protein sequence into a plasmid vector to be expressed in E.Coli as a fusion protein with GST. We also attempted to transform the normal ...
PrinceAladdin28's user avatar
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1 answer
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Will dissolved proteins pass through a 0.2 micron filter?

Given that there may be exceptions, can you usually expect protein to pass through?
ike9898's user avatar
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A question involving immunoprecipitation to identify interacting proteins?

Using recombinant Flag-tagged Dcr-2 and His-tagged protein X, pull-down assays were performed to determine whether protein X and Dcr-2 interact directly. The recombinant proteins (either alone or in ...
justbehappy's user avatar
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How is the volume of the dialysis buffer decided?

Is there a specific ratio between the volume of enzyme solution in semipermeable membrane bag and the volume of the buffer outside the membrane bag? I have been looking for a specific formula or ...
wswr's user avatar
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T7 Tagging Next to Met

Will a T7 tag still work if it is placed next to a start codon? Meaning, will it still work with a Met attached to it in the amino acid sequence? Thank you!
Kathryn's user avatar
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T7 Tagging in Synthetic Biology

What is a T7 tag and can if be used to purify synthesized proteins? Is it charge based like a His tag?
Kathryn's user avatar
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Can protein sample be made 2% Triton X-100 free?

The protein I am purifying needed an elution buffer with 2% Triton X-100. I formulated the elution buffer not keeping the CMC in mind. My goal is to make my protein sample triton free to check its ...
piscean_1993's user avatar
6 votes
1 answer
4k views

Can concentration of a protein be determined from a gel quantitatively (rough estimation)?

I've got a His-tagged protein in 6M urea, 500 mM imidazole buffer that needs to be quantified before dialysis to ensure there's enough protein worth dialysing. I ran out of my elution buffer which ...
piscean_1993's user avatar
3 votes
1 answer
484 views

How can I purify RNA after gel electrophoresis to remove residual acrylamide?

I sometimes use denaturing gel electrophoresis on a preparative scale to purify RNA produced by in vitro transcription. The major issue with this is that the sample after extraction from the gel still ...
user11595's user avatar
2 votes
1 answer
2k views

How does DNA resolve on size exclusion resin?

We generally have a good idea of how DNA separates using agarose gel electrophoresis, how well does DNA resolve on a SEC resin like superose? I get the impression that salt influences where it elutes. ...
bobthejoe's user avatar
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2 votes
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pI and pH relationship in context of ion exchange protein purification

I am confused about relationship between isoelectric point and pH in context of ion exchange protein purification. Why we cannot use this method for protein with pI below 7? Thank you very much for ...
Piesel's user avatar
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2 votes
1 answer
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How to identify active protein in a complex mixture?

I am trying to figure out how to identify which protein in a complex mixture is producing a certain effect. There is an assay for the effect, so anything (a fraction of the mixture) can be tested ...
Alex I's user avatar
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Removing bacterial contaminants from resin

We have a few Strep-tactin columns that had some growth in them and we would like to regenerate the columns back since the resin is quite expensive. Basic goal: remove the brown stuff. So far, I've ...
bobthejoe's user avatar
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4 votes
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Can I purify polyhydroxyalkanoates by heating the cells extensively?

Traditional methods of purifying polyhydroxyalkanoates (PHAs) and other bioplastics made by bacteria involve washing the cells with harsh chemicals or strong bases.I'm interested in maintaining the ...
LanceLafontaine's user avatar
4 votes
1 answer
561 views

How to wash the column during protein purification with GST tag?

I have been working with GST tagged proteins for the last 4 years and after loading the cell lysate into the column I was washing it with 20-30 column volumes of PBS and sometimes my proteins were ...
Engin Yapici's user avatar
5 votes
1 answer
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Using ion-exchange chromatography to purify DNA from a cell extract - Is DNA more negatively charged then RNA?

When applying this method we have a glass or plastic column of resin which is positively charged. Then we pour cell extract into the column in order to capture the negatively charged particles which ...
Emre's user avatar
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