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Questions tagged [purification]

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

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1answer
38 views

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 ...
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39 views

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 ...
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1answer
70 views

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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1answer
41 views

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-...
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0answers
39 views

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 ...
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1answer
272 views

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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2answers
176 views

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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0answers
28 views

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 ...
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1answer
77 views

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 ...
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2answers
376 views

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) ...
3
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2answers
280 views

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, ...
3
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1answer
109 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 ...
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1answer
116 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) ...
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1answer
51 views

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 ...
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4answers
4k views

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 ...
3
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1answer
445 views

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. ...
3
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1answer
1k 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 ...
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1answer
2k 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 ...
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1answer
2k views

Will dissolved proteins pass through a 0.2 micron filter?

Given that there may be exceptions, can you usually expect protein to pass through?
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1answer
304 views

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 ...
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0answers
1k views

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 ...
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2answers
47 views

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!
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1answer
1k views

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?
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0answers
366 views

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 ...
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1answer
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 ...
3
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1answer
354 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 ...
2
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1answer
1k 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. ...
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2answers
4k views

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 ...
2
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1answer
434 views

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 ...
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0answers
92 views

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 ...
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0answers
53 views

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 ...
4
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1answer
444 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 ...
5
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1answer
2k views

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 ...