#gel filtration

Chromatography

Correct elution order from Sephadex G-50?

Sephadex G-50 fractionates molecules within 1,500-30,000 Da range. Molecules above 30 kDa are fully excluded, cannot enter dextran pores, eluting at void volume first. Smaller molecules penetrate to varying extents; intermediate sizes partially included elute next, while very small molecules fully included elute last. Therefore progression from high to low molecular weight from early to late fractions defines correct order. Confusing small-to-large would invert physical principle. Recognizing large-to-small order aids in planning preparative separations, such as separating proteins from peptid

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Chromatography

Elute first in size exclusion chromatography?

Size exclusion chromatography separates strictly by hydrodynamic volume, not by charge or affinity. Porous inert beads allow small molecules to diffuse into pores, increasing path length and retention, while large molecules unable to enter are restricted to void space between beads. Consequently, large molecules travel shortest distance and emerge first, followed by progressively smaller species. This elution order opposite to many other methods enables molecular weight estimation, desalting, and removal of aggregates. Understanding inverse relationship between size and retention is crucial fo

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Chromatography

What is the exclusion limit of Sephadex G-50?

Sephadex is cross-linked dextran gel classified by G-value indicating degree of cross-linking and porosity. G-50 denotes moderate cross-linking with fractionation range for globular proteins approximately 1,500 to 30,000 daltons. Molecules larger than 30,000 daltons are excluded, eluting at void volume; smaller molecules penetrate and are retarded. Exclusion limit defines upper molecular weight threshold. Different grades like G-25, G-75, G-100 offer varying ranges. Knowledge of exclusion limits guides selection for desalting, buffer exchange, or separation of peptides from proteins, ensuring

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Chromatography

What is the Kd of a fully excluded solute?

In gel filtration, distribution coefficient Kd describes accessibility of solute to internal pore volume. It is defined by Ve = Vo + Kd*Vi, where Vo is void volume outside beads and Vi is internal pore volume. A solute completely excluded from pores cannot enter matrix, its elution volume equals Vo, implying Kd equals zero. A fully penetrating small solute exhibits Kd near one. Intermediate molecules show fractional values. Kd is never greater than one in ideal exclusion. Understanding Kd concept helps predict elution behavior, determine whether molecule is above exclusion limit, and evaluate

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Chromatography

In gel filtration, large molecules elute:

Gel filtration, also called size exclusion chromatography, uses porous beads with defined pore size. Separation is based on accessibility to internal pore volume. Large molecules exceeding pore diameter cannot enter beads and are limited to interstitial void volume, traveling fastest. Smaller molecules permeate pores, experiencing larger accessible volume and longer path. Consequently, large molecules elute first, followed by intermediate and small molecules. This inverse size-elution relationship allows desalting, molecular weight estimation, and protein oligomer purification without adsorpti

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

Which of the following is not a protein interaction method?

Techniques to map physical protein associations include co-immunoprecipitation that isolates native complexes via specific antibodies, fluorescence resonance energy transfer that detects proximity under 10 nm in live cells, phage display linking phenotype to genotype, yeast two-hybrid, surface plasmon resonance, and affinity pull-downs. SDS-PAGE is a fundamental analytical separation method that denatures proteins with sodium dodecyl sulfate and resolves them by molecular weight under electric field. It provides size information but alone does not report interaction without further methods lik

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Protein-Protein Interaction

In a research problem, a student finds proteins A-D with gel filtration, A-C with Co-IP, and B with Y2H. What is the bes

Gel filtration separates native protein complexes by hydrodynamic size; co-elution suggests residence in same large complex but does not distinguish direct versus bridged association. Co-immunoprecipitation from lysates preserves multiprotein assemblies, confirming co-complex membership. Yeast two-hybrid tests binary, direct physical interaction inside nucleus because reporter activation requires intimate contact between bait and prey fusions independent of other yeast proteins. When protein B uniquely appears positive in two-hybrid while others co-purify only, it indicates B is the direct int

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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