Gfp Purification
Gfp Purification - Related Videos
08:16
Plasmid Purification
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2023
Plasmid purification is a technique used to isolate and purify plasmid DNA from genomic DNA, proteins, ribosomes, and the bacterial cell wall. A plasmid is a small, circular, double-stranded DNA that is used as a carrier of specific DNA molecules. When introduced into a host organism via transformation, a plasmid will be replicated, creating numerous copies of the DNA fragment under study. In this video, a step-by-step generalized procedure is described for how to perform plasmid purification.
06:30
Gel Purification
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2023
Gel purification is used to recover DNA fragments after electrophoretic separation. DNA recovery from an agarose gel includes three basic steps: binding, washing and eluting from a silica column. DNA is believed to bind to silica in the presence of high salt via a salt bridge. Following binding, DNA is washed of impurities and eluted under low salt conditions disrupting this interaction. This video goes through a step-by-step, generalized procedure for cutting out a band from the gel, gel...
11:22
GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins
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Cited by 18 •
2013
In the present protocol, we demonstrate a highly efficient and cost-effective small-scale protein purification method, which allows purification of recombinant proteins by uniquely combining a cleavable GST-tag and a small His-tag.
15:09
Flow Cytometry-based Purification of S. cerevisiae Zygotes
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Cited by 5 •
2012
To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.
07:59
Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System
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Cited by 2 •
2016
We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.
FAQs
A polyhistidine tag provides a binding site for immobilized nickel or cobalt ions on the chromatography medium. The tagged GFP is retained while many untagged cellular proteins pass through or are removed during washing. Imidazole then disrupts this interaction and elutes the GFP, creating a selective enrichment step within the purification workflow.
Each chromatography strategy separates proteins using a different property. Affinity chromatography exploits a specific interaction, such as polyhistidine binding to nickel or cobalt. Charge-based methods distinguish proteins by electrical properties, whereas size-based methods separate them according to molecular dimensions. The selected approach therefore depends on which GFP property differs most usefully from cellular contaminants.
Fluorescence provides a functional readout that complements protein recovery. A purified GFP preparation can be examined for fluorescence while studying the protein’s folding and stability, rather than relying only on its physical isolation. Changes in fluorescence can therefore help connect purification results with the condition of the protein and its suitability for further experimentation.
The workflow begins by lysing cells to release GFP and other cellular proteins. Clarification removes insoluble material from the lysate before the sample enters chromatography. The separation step retains or resolves GFP according to the selected property, washing removes contaminants, and elution recovers the enriched protein for analysis or experimentation.
Washing improves sample quality by removing proteins that remain associated with the chromatography material without the desired binding strength. In a polyhistidine-tag system, imidazole is introduced to compete with the tagged protein for interaction with immobilized nickel or cobalt. This releases GFP from the medium and produces an eluted fraction for downstream use.
Purified GFP supports direct studies of protein expression, folding, fluorescence, and stability. In broader biological experiments, GFP also serves as a reporter for examining gene regulation, cellular localization, and molecular interactions. Its purification provides material for controlled analysis, helping researchers relate reporter behavior to the underlying protein and cellular process.