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Day 1

Buffer composition

• pH Buffer - how to choose the buffer
  • consider pKa, temperature effects on pKa, metal binding
  • counter ion, number of charges
  • concentration normally 10 to 40 mM
  • determine which form of the buffer to use, acid or base
  • consider expense and possible hazards
• Preparation
  • first, dissolve buffer solutes in solvent so that solution volume is 5-10% LESS than the desired final volume
  • add acid or base to adjust pH
  • "q.s. the volume" = bring the solution to desired FINAL volume (from Latin, quantum sufficit, "as much as suffices")
  • add other components (see list below)
  • confirm pH
• Additives
  • thiol reagents [dithiothreitol (DTT) = 1-5 mM, Beta-mercaptoethanol = 1:1000 dilution for 12 mM]
  • metal chelators (EDTA = 0.1 to 2 mM) NOTE: if histidine or imidazole is used for the buffer component, these compounds will also chelate heavy metals
  • water activity reagents [glycerol 20-30% v/v, sucrose 20-30% w/v (can use 50% for long-term storage)]
    • PERCENT means parts of one thing in 100 parts of total things present
    • for w/v, dissolve a weighed solute in a volume of liquid solvent such that the final volume = 100 parts
      remember the density of water
  • salts (range; ~0.15 M in mammalian cells)
  • proteins for stability [bovine serum albumin (BSA) = 0.1 to 10 mg/ml] *will not be adding today
  • protease inhibitors [metal chelators, specific inhibitors, phenylmethylsulfonyl fluoride (PMSF) 0.5 to 1 mM for serine proteases, pepstatin A, 0.0001mM for acid protease] *will not be adding today

Enzyme Assay

• need an assay in order to purify enzyme
• find most convenient assay method

Day 2

Cell lysis techniques: choice of starting material and storage conditions

• Select tissue or organism that is enriched in the target molecule.
• Determine if the starting material can be stored frozen before harvest or if it needs to be fresh.
• Determine how much material will be needed.
  • When large amounts of material are needed for studies such as crystallization or NMR, the protein may need to be cloned and produced in a culture to achieve gram quantities.

Cell disintegration and extraction

• Be familiar with different types of cell disruption techniques, particularly the methods used in our experiment. Familiarity means recognizing the name and having general understanding of the principle involved.
• Recognize there are limitations of the techniques.
  • Is the method capable of lysing the cells?
  • What is minimum and maximum size of sample that can be treated with the method?
  • Consider cost and availability of equipment.

Protein precipitation methods

• Why do proteins aggregate in aqueous solutions?
• Some of every protein will always be soluble - precipitation not good for dilute protein solutions.
• What are the available methods for precipitating proteins from solutions?
• What features make ammonium sulfate the chosen compound for precipitations?
  • solubility at 0-30 degrees Celsius
  • density (protein aggregates have density of about 1.33 g/cm3)
  • expense
  • multi-charged anion (cation is relatively unimportant)
• Common terms used in precipitations
  • percent saturated solution
  • fractionation or cuts
• How the precipitations are accomplished
  • Determine the amount of either solid or saturated solution to add to the existing solution from tables or calculations.
  • Add slowly with stirring to avoid high concentrations around undissolved material.

Day 3

Gel Filtration or Size Exclusion Chromatography

• Basic premise of method:
  • Sample is loaded onto the column and eluted with flowing buffer.
  • Fluid or buffer inside the beads is stationary and the mobile phase is the buffer flowing between and around the beads.
  • Only a certain percentage of the bead space is accessible to a given sized molecule; small molecules can access more space than large ones that do not fit through all the pores.
  • The more space of the bead a molecule has access to, the more time the molecule will spend inside the beads resulting in later elution from the column.
• Plates are the theoretical zones along column length in which a complete increment of exchange between bound and unbound molecules occurs. More plates equal more separation but can also increase band spreading.
• Resolution occurs when two or more components are separated to the extent of only 2% overlap or 98% of the component is pure.
• Parameters affecting separation on these columns:
  • Uniformity of packed bed (more uniform=better separation)
  • Flow rate of buffer (to a point, the slower the rate the better the resolution)
  • Length of column (longer columns give better resolution)
  • Concentration of protein solution (10-20 mg/ml)
  • Volume of sample loaded (should be 1-3% of column volume)
• Terms:
  • Bed volume (Vt)
  • Void volume (Vo)
  • Elution volume (Ve)
  • Flow rate (ml/min or ml/hr)
  • Linear flow rate (cm/hr)= flow rate (ml/hr) / cross sectional area of column (cm2)
• Choose column size based on predicted sample size.
  • column height should be about 20-40X the diameter.
  • Vt should be 30 -100X the sample size.
  • Estimate sample size from amount of protein in sample.
  
  

  Separation by Adsorption

  Adsorption refers to column material or support that bindscertain molecules.

  • ionic interaction - ion exchange chromatography, What amino acids are responsible for net charge at near neutral pH's? What is the maximum salt concentration that is usually needed to remove the proteins from DEAE columns? From CM columns? Consult the following tables for information for the selection of type of column and pH of buffer: 2nd ed. tables 5.4 and 5.5, 3rd ed. tables 6.3, 6.4, and 6.5. The ideal choice should buffer strongly and one component should be uncharged. What concentration is recommended and how is the choice of buffer made?
  • affinity - specific molecules covalently attached to support
    • dye - Molecules are hydrophobic and negatively charged. They appear to resemble nucleotides based on the types of proteins separated on these columns.
    • substrate or inhibitors - Substrates specific to the protein are attached to the column and hold the desired protein to be eluted with high salt or competitively with the same substrate.
    • hydrophobic - Hydrophobic residues are attached to the support. These residues interact with hydrophobic portions of the protein and these interactions are enhanced in high salt. Sample is usually loaded in high salt buffer and eluted by a decreasing salt gradient.

  Separation of components of the sample occurs through different affinities for the resin resulting in different retention times for the various molecules of the sample. The ability to separate two or more compounds is measured by the partition coefficient, alpha. Alpha is defined for each type of molecule and is the ratio of moving versus stationary molecules or in practice the fraction of protein adsorbed at any given time giving values between 0 and 1. The relative mobility of a type of molecule is Rf = (1-alpha) and is a crude estimate of the rate of travel through the column.

  Plates are the theoretical zones along column length in which a complete increment of exchange between bound and unbound molecules occurs. More plates equal more separation but can also increase band spreading.

  Resolution occurs when two or more components are separated to the extent of only 2% overlap or 98% of the component is pure.

  Read about factors of separation and resolution specifically applicable to size exclusion chromatography.
  Van Deemter equation from distillation theory: H=A / v+Bv+C
  v=flow rate
  A=diffusion effects that lead to band spreading
  B=non-equilibrium effects such as the exchange of solutes between the beads and the flowing solvent and between exchange sites
  C= column characteristics independent of flow rate and are fixed when the column is poured.

   

  Day 4

  Anion exchange

  Prepare for creating and running a salt gradient for elution from an anion exchange column. Use the instructions for the Q cartridge and past purifications (your library article and/or purifications of ADA) as a guide.

  What is the difference between a linear gradient and a step gradient? When is one preferred over the other? What methods are used to create gradients?

  Protein Determination methods

  Review the following four methods for protein determination and have a general understanding of the reaction causing the color change or principle of measurement. Review the sensitivity range for each.

  Bradford
  A280
  Lowry
  BCA-bicinconic acid

  Bradford (perform on crude extract on Day 2)- Currently the most popular method due to speed. Review Scopes or other books for theory of dye binding.

  Problems	Solutions
  Interfering buffer components	Perform dialysis or gel filtration to change buffer or precipitate protein with trichloroacetic acid
  Variation of color intensity developed by different proteins	Choose a standard protein that is most like the sample protein.
  Staining of glassware	Remove stain with ethanol rinses or 0.1 N HCl soaking.
  Staining of cuvettes leads to increasing background absorbance	Use clean cuvettes. Some staining results from proteins accumulating on the surface of the cuvette. Visually line up the sample tubes according to increasing "darkness" and sequentially obtain readings from "light" to "dark." There is no need to rinse the cuvette between reading when this method is employed. This method also gives a visual estimate of sample concentration and verifies that the samples are within the limits of the standard curve. Use disposable matched cuvettes. The solutions can be made directly in these. Be certain that the cuvettes are matched--variations up to 0.2 Abs have been noted in plastic cuvettes.

  
  
  

  A280-advantage is it's non-destructive to the protein.

  Problems	Solutions
  Interfering buffer components	Use dialysis or gel filtration to change buffer.
  Extinction coefficient is different for each protein	If known, use the extinction coefficient for the desired protein. If unknown, use 1 ml/mg*cm, which is near the median for known proteins.
  Other absorbing substances present, such as DNA and RNA in crude homogenates	Remove DNA by streptomycin or polyethylenimine precipitation. Use the ratio of 260 to 280 to correct for presence of nucleic acids.

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  Copyright, Acknowledgements, and Intended Use
  Created by B. Beason (bbeason@rice.edu), Rice University, 9 June 1999
  Updated 3 January 2012