Fplc Purification
Fplc Purification - Related Videos
07:58
Electroplating of Thin Films
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2023
Source: Logan G. Kiefer, Andrew R. Falkowski, and Taylor D. Sparks, Department of Materials Science and Engineering, The University of Utah, Salt Lake City, UT Electroplating is a process that uses electric current to reduce dissolved metal cations so that they form a thin coating on an electrode. Other thin film deposition techniques include chemical vapor deposition (CVD), spin coating, dip coating, and sputter deposition among others. CVD uses a gas-phase precursor of the element to be...
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...
10:08
Fabrication of Large-area Free-standing Ultrathin Polymer Films
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Cited by 10 •
2015
We describe a method for the fabrication of large-area (up to 13 cm diameter) and ultrathin (as thin as 8 nm) polymer films. Instead of using a sacrificial interlayer to delaminate the film from its substrate, we use a self-limiting surface treatment suitable for arbitrarily large areas.
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.
FAQs
The separation mode should match the property that distinguishes the target from other sample components. Affinity chromatography uses differences in binding, ion-exchange chromatography separates according to charge, and size-exclusion chromatography separates according to molecular size. Selecting among these modes allows a bioengineer to tailor the purification strategy to the biomolecule and the composition of the starting sample.
Elution depends on how strongly each sample component interacts with the selected stationary phase. Components with different affinity, charge, or molecular size move through the column differently and therefore appear at different points in the run. The detector records signals such as ultraviolet absorbance, helping identify when separated material exits and which fractions may contain the target.
Buffered conditions help maintain an environment that preserves biological activity while the sample passes through the column. This consideration is especially important when the purified biomolecule will support biochemical, structural, or therapeutic research. Automated pumping and signal monitoring further provide a consistent workflow for tracking separation without relying only on manual handling of the sample.
A typical workflow places the sample in a system where a pump moves it through a column containing the selected stationary phase. As components separate, detectors monitor signals such as ultraviolet absorbance. The system collects the emerging material in separate fractions, which can then be examined individually or combined when they contain the desired purified biomolecule.
Collected fractions provide discrete portions of the column output for downstream evaluation. Researchers can analyze their detector signals or other available measurements to determine which portions contain the desired material, then pool compatible fractions. This approach converts the continuous separation process into selected sample portions suitable for later biochemical, structural, or therapeutic research.
Bioengineers use this approach when they need defined biomolecules for further investigation or processing. Supported applications include purifying recombinant proteins, processing antibodies, and preparing materials for structural and biochemical studies. The same capability also contributes to therapeutic research, where preserving biological activity during purification can be important for evaluating the resulting biomolecule.