In both experiments, affinity purification and ion exchange (IEX) columns, we have loading, washing, and elution steps. Please describe and explain the purpose for each steps.
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- When eluting your analyte of interest using HPLC, gradient elution can be used. How does Gradient elution differ from Isocratic and comment on the advantages of using this type of mobile phase elution?In nickel column affinity chromatography，how does the protein binds to column？How is the protein eluted from the column？What are the advantages of gel filtration as a technique for protein purification? Identify two types of gels (sieving matrix) that may be used for gel filtration chromatography and discuss the types of samples which may be used for each. Discuss the principle behind affinity chromatography and differentiate it from gel filtration chromatography.
- ) In urea assay (Catalog #K375-100) what is the nmol (range) it can detect?Why are buffers important in biochemical experiments?What mode of chromatographic separation (RPLC, NPLC, HILIC, SEC, IEX) is best used for the specified Experiment Analysis of monosodium glutamate from Beef Broth Cubes, including those of other ions like NaCl? What stationary phase and mobile phase (together with elution technique), and other chromatographic conditions needed?
- Gel-filtration chromatography is a useful method for removing salts, such as ammonium sulfate, from protein solutions. Describe how such a separation is accomplished.What is doping? Explain the mechanism of mass spectrometry.Saturation experiments using radioligand is one method of determining the affinity of a ligand to a drug target. What are other methods used in high-throughput screening (HTS) of compound libraries that do not use a radioligand?
- What is your overall dilution factor if you complete 3 serial dilutions using a 100-fold dilution each time? (Show your work)The protein concentration of a known standard is 100mg/mL If you prepared a serial dilution, mixing 10μL of protein with 40μL of water what would be concentrations of the first 3 dilutions?Total protein: Do you think that refractive index could be used as a regular method for serum protein determinations? If yes, what limitations does it have. If not, why not? What are the major interferences in the biuret method? Refractometry method?