Assays

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Assays
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Overview

• Assay types
• Protein assays
• Activity assays
• Specific Activity
• Yield
• Purification Factor
• Summarizing the results of the purification

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Review

• Review of spectrophotometry and standard curves
• Seidman and Moore, Basic Lab Methods for
  Biotechnology.

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What is an assay?

• An assay is a method used to measure something
• The something is usually not visible
• Example
• Measure the amount (how much) of DNA you have

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What is an assay?

• How can you do that?
• UV absorbance at 260
• Diphenylamine assay (colorimetric assay)
• DNA reacts with a chemical to form a colored
  product
• Measure the amount of colored product by the
  amount of light that is absorbed at a particular
  wavelength.

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How do we measureproteins?

• By how much is there
• Mass
• Weight
• Concentration
• By what they do
• Activity

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Colorimetric AssaysStandard Curves

• Light Absorption
• Spectrophotometer
• Absorbance of a substance depends on
• Wavelength
• Path length
• Concentration of substance
• Nature of substance

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Colorimetric AssaysStandard Curves

• Beer-Lambert Law
• A Ebc
• A Absorbance
• b path length
• E constant
• c concentration

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E constant Absorbtivity Constantor Extinction
Coefficient
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Protein Assays

• UV absorbance -quick, non-destructive
• Monitoring column fractions

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Protein Assays

• Colorimetric assays
• More accurate than UV
• Several types
• Biuret
• Lowry
• Bradford (Bio-Rad)
• BCA (Pierce)

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ChooseTotal Protein Assay

• Sensitivity- Lowry or Bradford
• Interfering substances
• If detergents - use BCA
• Ease
• One reagent
• Linearity varies

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Bio-Rad Assay

• Based on the Bradford assay
• Measures the amount of protein (any and all
  proteins)
• Based on a dye, Coomassie Brilliant Blue G250
• Binds to basic and aromatic amino acids
• Absorbance Maximum shifts from 465 nm
  (brownish)to 595 nm (blue) when binds protein

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Colorimetric AssayStandard Curve

• Take a series of standards
• Add dye
• Graph amount of protein vs. O.D. for the
  standards
• Take unknown and measure O.D.
• Which protein do you use for standard?
• Target protein Pure ß-galactosidase
• Bovine Serum Albumin - generic protein

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Bio-Rad Assay
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How will you know youve purified a specific
protein?

• Need an assay that detects target and only the
  target
• Activity assays

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Specific Assays forTarget Protein

• Unique identification of target protein
• Is protein colored?
• What kind of protein?
• Enzyme?
• Is there an antibody?
• How can you measure its activity?

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An Enzyme IsA Catalyst
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Enzymes

• Substrate(s) product(s)
• Types of Enzyme Assays
• Measure product formation
• Measure consumption of substrate
• Measure consumption of cofactors

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Enzymes

• Method of Detection
• Colorimetric assay
• Spectrophotometer
• Radiometric assay
• Radioactive tag

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Not All ProteinsAre Enzymes

• Other activity assays
• Signaling protein for cell cycle
• Cell adhesion molecule
• Transporting protein

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ß-GalactosidaseAssay

• ß-galactosidase catalyzes the following reactions
• Lactose glucose galactose
• ONPG ONP galactose

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ß-GalactosidaseAssay lactose as substrate
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ß-GalactosidaseAssay

• Substrate is ONPG
• Product is ONP
• ONP is yellow so can monitor the change in amount
  of yellow color using VIS Spec at 410-420nm
• Measure formation of colored product

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ONPG as substrate
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Preliminary Activitiesfor Enzyme Assays

• Determine the Extinction Coefficient of ONP
• Do a standard curve with different concentrations
  of ONP

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Preliminary Activitiesfor Enzyme Assays

• Or, could use published value
• 4500M-1cm-1
• We will do this
• If 1 M has A of 4500, then what s are in the
  linear range of the spec?
• Spectrophotometer reads from 0 to 2 absorbance
  units

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Enzyme Assay

• Colorimetric assay based on Beers law
• A Ebc
• E Extinction Coefficient
• Also called absorbance constant
• Absorbtivity constant
• Can determine concentration by measuring
  absorbance

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Enzyme Assay

• How do you get Absorbance constant?
• Do standard curve (its the slope)
• Published value

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y mx b
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• So for this assay, we want to convert the of
  ONP into units of enzyme. How do we relate
  concentration to units?
• Definition of a unit?
• The amount of enzyme that will hydrolyze 1
  µmole/minute of ONPG at 37C
• So the equation is
• Units of ß-gal A420 x 0.380
• minutes at 37C

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Enzyme AssayProcedure

• Add 10-50µl of sample (containing enzyme) to 1 mL
  of Z buffer
• Add 0.2 mL of ONPG (substrate) at 4 mgs/ml
• Let reaction develop - should see pale yellow
  color in about 10-15 minutes
• Add 0.5mL of Stop solution and read on Spec at
  420 nm

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Enzyme AssayProcedure Cont.

• What is your negative control?
• Need it to calibrate the spectrophotometer
• Need to get an absorbance of 0.3-0.9 in 10 min or
  greater. If a bright yellow color develops
  quickly, start over with more dilute sample.
• Why?

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Sample Calculation
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Yield
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So You Know The Yield

• OK- that tells you how much is there BUT
• What tells you how pure it is?

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MonitoringProtein PurificationSpecific
Activity

• One measure of purity
• Specific Activity measures ratio of target
  protein to all protein
• So need two assays
• One for target protein
• ß-gal assay
• One for all proteins
• Bio-rad assay

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Bio-Rad Assay
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Specific Activity

• Specific Activity Target protein activity
• Total protein mass
• Specific Activity units/ml
• mg/ml total protein
• As protein becomes more pure, contaminants
  decrease, lowering the amount of total protein
• Target protein will decrease slightly but the
  ratio of target to total will increase

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Lab Manual
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Monitoring The Purification
Dark bars Mass of Total Protein Light bars
Activity of Target protein
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Purification Factor

• Also called Enrichment or Fold Purification
• Purification Factor
• How much more pure is it than when you started?
• Specific Activity of this Step
• Specific Activity of Crude Extract

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Purification Factor (Enrichment)
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Monitoring the Purification
Dark bars Mass of Total Protein Light bars
Activity of Target protein
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Real Data From Student Project
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E.Coli Cell Paste
Cell disruption
Pellet
Centrifugation
Crude Extract (Supernatant)
Aliquot
Ammonium Sulfate Precipitation
Centrifugation
Supernatant
Aliquot
Pellet (Resuspend)
Dialysis
Pellet
Centrifugation
Dialysate
Aliquot
Ion Exchange Chromatography
Collect fractions- assay
Assays for Specific Activity
SDS PAGE Western Blotting
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ß-GalactosidaseCharacteristics

• Molecular weight is 518,000 daltons
• Composed of 4 subunits (tetramer)
• Monomer is 130K
• Fairly heat stable in presence of
  ß-mercaptoethanol or DTT and 0.01M MgCl2
• 50 loss of activity after 40 minutes
• Without ß-ME, all activity lost after 10 minutes

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ß-GalactosidaseCharacteristics

• Stable from 6-8 pH
• Isoelectric point (pI) is 4.6