Polymeric Cryogel‐Based Boronate Affinity Chromatography for Separation of Ribonucleic Acid from Bacterial Extracts

Akhilesh Kumar Shakya1, Akshay Srivastava2, Ashok Kumar3

1 Department of Chemical Engineering, Texas Tech University, Lubbock, Texas, 2 Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Republic of Ireland, 3 Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur (U.P)
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 10.16
DOI:  10.1002/0471142700.nc1016s63
Online Posting Date:  December, 2015
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Abstract

Three‐dimensional monolithic columns are preferred stationary phase in column chromatography. Conventional columns based on silica or particles are efficient in bioseparation though associated with limitations of nonspecific interaction and uneven porosity that causes high mass transfer resistance for the movement of big molecules. Cryogels as a monolith column have shown promising application in bioseparation. Cryogels column can be synthesized in the form of a monolith at sub‐zero temperature through gelation of pre‐synthesized polymers or polymerization of monomers. Cryogels are macroporous and mechanically stable materials. They have open interconnected micron‐sized pores with a wide range of porosity (10‐200 μm). Current protocol demonstrated the ability of poly(hydroxymethyl methacrylate)‐co‐vinylphenyl boronic acid p(HEMA‐co‐VPBA) cryogel matrix for selective separation of RNA from the bacterial crude extract. © 2015 by John Wiley & Sons, Inc.

Keywords: cryogel; RNA separation; monolithic column; boronate‐affinity chromatography

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Poly(Hydroxyethylmethacrylate‐co‐Vinylphenylboronic Acid) [Poly(Hema‐co‐VPBA)] Cryogel
  • Basic Protocol 2: Preparation of Bacterial Crude Extract for RNA Isolation
  • Basic Protocol 3: Boronate‐Affinity Cryogel Column Chromatography to Isolate Bacterial RNA
  • Basic Protocol 4: Selectivity of Poly(Hema‐co‐VPBA) Cryogel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Poly(Hydroxyethylmethacrylate‐co‐Vinylphenylboronic Acid) [Poly(Hema‐co‐VPBA)] Cryogel

  Materials
  • 6% (w/v) 2‐Hydroxyethyl methacrylate (HEMA; Sigma Aldrich) monomer solution in degassed water (see recipe)
  • 1% (w/v) 4‐Vinylphenylboronic acid (VPBA; Sigma Aldrich) monomer solution in degassed water (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • Polyethylene glycol diacrylate (PEGDA; Sigma Aldrich): 3.5% (v/v)
  • Nitrogen
  • Ammonium persulfate (APS; Sigma Aldrich): 0.1% (w/v) solution in degassed water (see recipe)
  • N,N,NN’‐Tetramethylethylenediamine (TEMED; Sigma Aldrich, Germany): 0.1% (v/v)
  • 5‐mL syringes
  • Stainless steel test tube stand
  • Cryostat (Julabo USA) with methanol solvent
  • Peristaltic pump
  • Freeze dryer, optional

Basic Protocol 2: Preparation of Bacterial Crude Extract for RNA Isolation

  Materials
  • E. coli Bacterial strain
  • LB broth medium (see recipe)
  • Tris·Cl buffer, pH 8.0
  • Loop or sterile toothpick
  • Shaking incubator (Thermo Fisher Scientific)
  • 50‐mL Falcon tubes
  • Centrifuge
  • Soni prep 150 plus (MSE, United Kingdom Limited)

Basic Protocol 3: Boronate‐Affinity Cryogel Column Chromatography to Isolate Bacterial RNA

  Materials
  • Binding buffer (see recipe)
  • Bacterial crude extract (from protocol 2): always store the bacterial crude extract at 4°C
  • Elution buffer (see recipe)
  • Synthesized poly(HEMA‐co‐VPBA) and pHEMA cryogel (as a control) columns in dimensions of 2.5 × 1.3 cm (Fig. A,B; as prepared in protocol 1)
  • Peristaltic pump (Bio‐Rad)
  • Spectrophotometer

Basic Protocol 4: Selectivity of Poly(Hema‐co‐VPBA) Cryogel

  Additional Materials (also see protocol 3)
  • DNase enzyme (Invitrogen): Reconstitute lyophilized DNase in DNase buffer (100 mM Tris·Cl, pH 7.5, 25 mM MgCl 2, 5 mM CaCl 2) and store up to 6 to 12 months at −20°C
  • RNase enzyme (Invitrogen): Reconstitute lyophilized RNase in RNase buffer (5 mM EDTA, 300 mM NaCl, 100 mM Tris·Cl pH 7.5)
  • 0.1 M Formic acid
  • Agarose (Sigma Aldrich): Dissolve 1% (w/v) agarose in 1× Tris‐acetate‐EDTA (TAE buffer)
  • Whole yeast RNA and DNA powders (Sisco Research Laboratories Limited): As controls in agarose gel electrophoresis
  • 1× TAE buffer (see recipe)
  • 6× sample loading buffer: Dissolve 1 mL sterile H 2O, 1 mL glycerol, and 0.05 mg of bromphenol blue dye and store the buffer in frozen conditions for long‐term storage
  • Agarose gel electrophoresis unit (Bio‐Rad)
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Figures

Videos

Literature Cited

Literature Cited
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