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1
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1answer
133 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
71 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?
1
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1answer
35 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
65 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 ...
2
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1answer
89 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?
1
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2answers
31 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!
2
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0answers
165 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 ...
4
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1answer
641 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
votes
1answer
147 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 ...
3
votes
1answer
205 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 ...
2
votes
1answer
245 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. ...
2
votes
2answers
626 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
votes
1answer
130 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 ...
5
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1answer
1k 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 ...
2
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0answers
77 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 ...
3
votes
0answers
40 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 ...