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实验方法> 生物信息学技术> 数据库>Size‐Exclusion Chromatography with On‐Line Light Scattering

Size‐Exclusion Chromatography with On‐Line Light Scattering

关键词： size exclusion chromatography来源： 互联网
  • Abstract
  • Table of Contents
  • Materials
  • Figures
  • Literature Cited

Abstract

 

This unit describes the use of size?exclusion chromatography with on?line light scattering, UV absorbance, and refractive index detectors (SEC?LS/UV/RI) to determine: (a) the molecular weight of simple proteins containing no carbohydrates, (b) the molecular weight of glycoproteins, and (c), most importantly, the molecular weight and stoichiometry of protein?protein complexes or protein?carbohydrate complexes. Multiangle light scattering is also discussed.

     
 
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Table of Contents

  • Basic Protocol 1: Using Refractive Index and Light Scattering to Calculate the Molecular Weight and Degree of Self‐Association of Proteins Containing no Carbohydrates (Two‐detector Method)
  • Alternate Protocol 1: Combination of LS and UV Detectors to Calculate the Molecular Weight of Nonglycosylated Proteins (Two‐detector Method)
  • Alternate Protocol 2: SEC/LS for Large Proteins: Debye Analysis
  • Alternate Protocol 3: Absolute Molecular Weight Calibration Method
  • Basic Protocol 2: Calculating the Stoichiometry of a Protein‐Protein Complex Containing no Carbohydrates Using LS/RI
  • Alternate Protocol 4: Calculating the Stoichiometry of Protein‐Protein Interactions for Nonglycosylated Proteins Using LS/UV
  • Basic Protocol 3: Calculating the Molecular Weight of Glycoproteins or Protein Conjugates Using Three Detectors
  • Basic Protocol 4: Determining the Stoichiometry of a Protein‐Protein Complex Containing Carbohydrates
  • Support Protocol 1: Sample Analysis: Receptor‐Ligand Interactions
  • Basic Protocol 5: Analysis of Protein‐heparin Interactions Using Three Detectors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Using Refractive Index and Light Scattering to Calculate the Molecular Weight and Degree of Self‐Association of Proteins Containing no Carbohydrates (Two‐detector Method)

  Materials
  • Protein solutions of unknown molecular weight, at ∼1 mg/ml
  • recipeCalibration standards (also see recipe ): ∼1 mg/ml ribonuclease, ovalbumin, and BSA in column buffer
  • recipeElution (column) buffer (see recipe and unit 8.3 )
  • Size‐exclusion (gel‐filtration) chromatography column (unit 8.3 )
  • HPLC apparatus (units 8.3 & 8.7 ) including light‐scattering (miniDawn, Wyatt Technology) and refractive index (Agilent 1047A) detectors
  • Additional reagents and equipment for gel‐filtration chromatography (unit 8.3 ) and HPLC (unit 8.7 )
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Figures

  •   Figure 20.6.1 A typical configuration for SEC‐LS/UV/RI (size‐exclusion chromatography with on‐line light scattering, UV absorbance, and refractive index detection).
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  •   Figure 20.6.2 A typical plot of LS/RI versus the molecular weights of protein standards (RNase, ovalbumin, and BSA). 100 µl of each protein standard was injected onto a SEC column separately, to obtain more accurate data (slight overlap may occur for some SEC columns). Typical protein concentrations were 2.0, 1.5, and 1.5 mg/ml for RNase, ovalbumin, and BSA, respectively. Reproduced from Wen et al. () with permission of Academic Press.
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  •   Figure 20.6.3 Chromatogram of BSA that contains monomers, dimers, and other oligomers. Commercial BSA from Sigma is a mixture of monomers, dimers, and higher oligomers. The molecular weight of peak 1 calculated from the two‐detector method is 132,000, which agrees well with twice the BSA sequence molecular weight of 66,269, indicating that peak 1 is a BSA dimer. 100 µl of 4 mg/ml BSA was injected onto a Superose 6 column (Pharmacia) with PBS as eluent at 0.5 ml/min flow rate. The solid line is the LS signal and the dashed line is the RI signal. Reproduced from Wen et al. () with permission of Academic Press.
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  •   Figure 20.6.4 Chromatograms of native and reduced carboxymethylated (RCM)–RNase. 100 µl of each protein was injected onto a Superdex 75 column (Pharmacia) with PBS as eluent at a flow rate of 0.5 ml/min. (A ) 1 mg/ml of native RNase; (B ) 1 mg/ml of RCM‐RNase; (C ) chromatograms in panels A and B are put onto the same scale for comparing the LS/RI ratios of native and reduced RNase. The lines are the same as defined in Figure . Reproduced from Wen et al. () with permission of Academic Press.
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  •   Figure 20.6.5 Chromatograms of TNF, sTNFR, and the mixture of sTNFR and TNF. 100 µl of each protein was injected onto a Superose 12 column (Pharmacia) with PBS as eluent at a flow rate of 0.5 ml/min. (A ) TNF control sample (no sTNFR); (B ) sTNFR control sample (no TNF); (C ) a mixture of sTNFR and TNF made at a molar ratio of about three sTNFR per TNF. The solid line is the LS signal, the dashed line is the RI signal, and the dotted line is the UV signal. Reproduced from Wen et al. () with permission of Academic Press.
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  •   Figure 20.6.6 Chromatograms of E. coli SCF. CHO SCF contains >30% carbohydrate and thus the three‐detector method was used. 100 µl of 3.8 mg/ml of E. coli SCF was injected into a Superdex 200 column (Pharmacia) with PBS as eluent at a flow rate of 0.5 ml/min. The solid line is the LS signal, the dashed line is the RI signal, and the dotted line is the UV signal. Reproduced from Wen et al. () with permission of Academic Press.
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  •   Figure 20.6.7 Chromatograms of sTrkB and the mixture of sTrkB and BDNF. 100 µl of each protein was injected onto a Superdex 200 column (Pharmacia) with PBS as eluent at a flow rate of 0.5 ml/min. (A ) sTrkB control sample (no BDNF); (B ) a mixture of sTrkB and BDNF made at a molar ratio of about two sTrkB per BDNF. The lines are the same as defined in Figure . Reproduced from Wen et al. () with permission of Academic Press.
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  •   Figure 20.6.8 Chromatograms of MAb35, sHer2, and the mixture of sHer2 and MAb35. 100 µl of each protein was injected onto a Superdex 200 column (Pharmacia) with PBS as eluent at a flow rate of 0.5 ml/min. (A ) MAb35 control sample (no sHer2); (B ) sHer2 control sample (no MAb35). (C ) A mixture of MAb35 and sHer2 made at a molar ratio of about 2.7 sHer2 per MAb35. The lines are the same as defined in Figure . Reproduced from Wen et al. () with permission of Academic Press.
    View Image
  •   Figure 20.6.9 Chromatograms of bFGF and HMWH mixtures (UV absorbance traces only). All samples contained 117 µM bFGF, and the amount of HMWH varied. The [HMWH]/[bFGF] molar ratio was 0.011:1 for B, 0.022:1 for C, 0.056:1 for D, 0.11:1 for E, 0.22:1 for F, and 0.43:1 for G. Sample A (bFGF control) is not shown here. 100 µl of each mixture was injected onto a Superdex 200 column (Pharmacia) with PBS as eluent at 0.5 ml/min flow rate. Reproduced from Wen et al. () with permission of Academic Press.
    View Image

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Literature Cited

Literature Cited
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