BISC 220 Lab 2 by ert554898

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									  BISC 220 Lab 2

Protein Purification
         by
      Affinity
 Chromatography
         &
 Determination of
 Specific Activity
          TO DO TODAY
• Extract protein induced last week from the
  bacterial cells
   – chemical cell lysis with“Bacterial Protein
     Extraction Reagent”- DETERGENT
   – Removal of cell debris by centrifugation
• Partial Purification of protein
   – Separate the protein of interest from other
     proteins by Affinity Chromatography
• Assay the total protein in the CRUDE
  EXTRACT & the PURIFIED
  FRACTION and Assay the specific
  activity of the b-gal in each.
• Quantify & compare total protein &
  specific activity for both fractions & then
  calculate % yield & purification factor
  Affinity Chromatography:
    the General Strategy
                 Specific         Elution
                 binding         (release)




                 Can use altered pH, salt,
                 competitor molecule for
                 elution.
Example:
 Protein Purification via Metal
Chelate Affinity Chromatography




 • The 6xHis tag on the b-gal will bind tightly
   to the Ni+ agarose, which can be
   separated from the supernatant by
   centrifugation.
 • Other proteins that interact non-specifically
   (weakly) with the Ni+ agarose will be
   removed during washes.
 • The 6xHis b-gal will be eluted (released)
   from the beads by competition with
   imidazole (a molecule similar to histidine).
Imidazole & histidine: a
 structural comparison




 Imidazole
   ring      • Excess imidazole out-
               competes 6xHis-b-gal for
               binding to Ni+ agarose
        b-Gal Partial
    Purification Protocol:
    Things to Remember
• Follow directions carefully
• Know where you are in process
• Mix well, measure carefully &
  don’t confuse reagents
• Be careful making dilutions
• End up with 2 fractions to assay:
   – Crude Extract-CE
   – Purified (partially) Fraction-
     contains b-galactosidase-PF
  Determining Total Protein
Content Spectrophotometrically
        (both CE and PF)




          Free Coomassie
             Blue Dye
        (Bradford Reagent)
       Absorbance at 470 nm


                  + Protein


       Dye Bound to Protein
       Absorbance at 595 nm
              Making a Standard Curve from
              Absorbance readings of known
             BSA concentrations using Linear
                       Regression
Absorbance




                      Concentration (mg/ml)



   • y =m x + b, where m is the slope & b is the y-
     intercept. Use the equation generated by Excel to
     solve for x (concentration)

   • Will need to dilute a 1 mg/ml BSA stock to make
     0.1, 0.2, 0.4, 0.6 & 0.8 mg/ml samples (not a
     dilution series; make 200 µl of each dilution)
       Measuring the Specific
         Activity of b-gal




            Colorless



• ONPG = artificial substrate for b-gal

• Specific activity = Vmax (maximum velocity) =
  rate of appearance of product under conditions
  of saturating amounts of substrate
  (mmol/min/mg protein)

• Detect appearance of ONP (product) by
  absorbance at 420 nm.
  What does the Specific
   Activity tell you?

• With greater purification of an
  enzyme, total activity & % yield
  will decrease but specific activity
  will increase.

• Purification factor = ratio of
  specific activities of PF/CE; a
  higher purification factor means
  that more of the protein in the
  sample is the enzyme of interest.
Calculating the Concentration of
  ONP from A420 Readings of
       Enzyme Reactions
                                  A
     Beer-Lambert Law:        C=
                                 e*l
C = concentration (moles/L)
A = absorbance reading at given wavelength (no
    units)
e = molar extinction coefficient at given l
    (M-1cm-1)
l = spectrophotometer path length (cm)

           e for ONP = 4800 M-1cm-1

• Must use an amount of enzyme that produces
  an amount of product (in a defined reaction
  time) that gives an absorbance reading in the
  reliable range for the spectrophotometer (0.1-
  1.0). Will try several dilutions of CE & PF.
      Making Dilutions
      V1 x C1=V2 x C2
• FOR PROTEIN ASSAY
 1. BSA stock 1mg/ml
    Working dilutions (want 200µl):
    0.1, 0.2, 0.4, 0.6, 0.8 mg/ml
 2. Purified (PF) & Crude Extract (CE)
    1:5 dilution with Z buffer (want 300 µl)

• FOR ENZYME ASSAY
 1. Purified Fraction
   Want 250µl each of 1:100, 1:200, 1:400&
   1:800 dilutions
 2. Crude Extract
    Want 250 µl each of 1:50, 1:100, 1:200 &
    1:400 dilutions
 (Use serial dilution strategy for these.)
Things to Remember about
         Assays:
Protein Assay
• Make 2 reagent blanks instead of
  1 since using double beam
  spectrophotometer- 11 tubes
• Mix dilutions well & keep on ice
• Timing is not critical
b-Gal Assay
•   Keep all diluted fractions on ice
•   Make 2 reagent blanks instead 1
•   Timing IS critical!!!
•   You will have a lot of tubes in
    your ice bucket. Be VERY
    careful not to mix things up—
    label well.
         Before You Leave
• Add glycerol to remaining purified fraction &
  give to instructor to freeze
• Give 3 samples to instructor (properly
  labeled—see p. 41)
• Clean up your work area



              Homework
• Complete calculations & answer questions on
  p. 42
• Don’t wait until the last minute to do the
  calculations!
• When calculating protein concentrations, don’t
  forget to account for the dilutions.
• For other calculations, follow examples on p.
  48-50.

								
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