Column Directions

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Column Directions
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Basic Idea

• Add the protein sample (after dialysis) to the
  column
• Some of the proteins will bind to the column
  because they are negatively charged at this pH

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Basic Idea

• Some of the proteins will not bind and will pass
  through the column
• After all the unbound proteins have been washed
  through, we will increase the salt concentration
  in order to selectively elute (desorb) the
  proteins which are bound
• One of these is ß-galactosidase

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Six Phases

• Set-up column equipment
• Prepare resin and pour column
• Loading
• Washing the unbound through
• Elution
• Regeneration

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Column Set UpEquipment Check

• Connect a beaker of DI water to the pump.
• Connect in series pump into column, into UV
  monitor into fraction collector chart recorder
  is connected to UV monitor
• Find the on/off switches (often on the bottom)
• Check that you know what the buttons do
• Chart recorder 1 V
• Use the event marker button on the UV monitor to
  mark the chart recorder

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Materials Needed

• Column
• DEAE Sepharose
• Equilibration Buffer NTM Buffer
• 0.2M NTM and
• 0.2M NTM with 10X TRIS
• Elution Buffer
• 0.5M NTM

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Materials NeededCont

• Protein sample in 0.2M NTM
• Chromatography Set up
• Pump
• UV Monitor
• Fraction Collector
• Chart Recorder

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ChromatographySet up
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To Do

• Get familiar with equipment
• Calibrate pump or check existing calibration
• Set Fraction Collector on drops
• Know how many drops in a mL
• Fraction collector counts drops
• Learn to work chart recorder
• Sensitivity
• Zero the UV monitor on 0.2M NTM

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To Do Cont

• Make sure that UV monitor communicates with chart
  recorder
• Find the event marker
• Standardize settings
• 1.0 AUFS on UV monitor
• 5 mm/min chart recorder speed

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Next

• Pour the column
• Make slurry of resin and 0.2M NTM buffer
• Pour it in one even motion into column
• Let settle

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Next

• Equilibrate the column
• Pack the column at 1 ml/min
• 2 stages
• 1. 10X Tris until pH out pH in
• 2. 0.2M NTM for 2 column volumes

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Running TheColumn

• Load the sample on column
• Wash the column with 0.2M NTM
• Continue running buffer through column until all
  unbound proteins are washed through
• Until the O.D. is below 0.1

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Running TheColumn Cont

• Start the elution phase
• Connect gradient of 0.2M NTM to 0.5M NTM buffer
• Collect fractions using fraction collector or
  manual
• Store on ice after each fraction collected
• Assay fractions using plate reader

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Elution
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Important

• Dont connect the gradient maker (elution phase)
  to the column until unbound is washed through
• Look carefully at buffer - dont mix them up.
• What would happen if you dialyzed your sample
  against 0.5M NTM buffer
• How could you fix it?

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How to AssayColumn Fractions

• Plate reader-
• Simple spec for individual wavelengths - reads A
  for 96-well plates
• Plate reader assay
• LM page 62
• Well A-1 is blank

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Lower Tech Alternatives

• Gravity feed columns
• Read fractions on a regular spec
• Gradient makers
• Make your own or
• Use steps instead of gradient