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Protein Purification Lab C2 Pages 101 to 142

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Protein Purification Lab C2 Pages 101 to 142 Lab C.2 Four Periods Protocol Page 118-142 Be sure to read theory starting page 104 ... – PowerPoint PPT presentation

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Title: Protein Purification Lab C2 Pages 101 to 142


1
Protein Purification Lab C2Pages 101 to 142
  • Lab C.2
  • Four Periods
  • Protocol Page 118-142
  • Be sure to read theory starting page 104

2
Exam
  • Exam March 12
  • Includes Carbohydrates, Enzyme kinetics, and all
    protein labs and material related there to.
  • Pay attention to the powerpoints
  • Read theory sections in the lab manual
  • Will be about one hour in length
  • Example of exam with answers is posted on web

3
You Have
  • Become skilled at using micro pipetters
  • Have learned to use the spectrophotometer
  • To determine concentration of an unknown
  • Beers Law
  • To measure activity of an enzyme
  • Have learned how to organize experimental
    protocols
  • Have learned how to prepare a report.

4
In the next days
  • You will use all of these skills to perform a
    fundamental exercise in Biochemistry/Molecular
    Biology
  • Will learn basic protocols in protein
    purification and analysis

5
Protein Purification
  • A black art (proteins have personality)
  • Requires knowledge of protein
  • What kind of cell is it coming from
  • What part of cell
  • What does it do
  • Particularly helpful
  • Size
  • Composition

6
Strategy
  • Move from organism to pure protein in as few
    steps as possible with as little loss of activity
    (assayable quality) as possible
  • Time and temperature are factors

7
Protocols for Protein Purification
  • Highly individualized
  • Use a common approach
  • Fractionate crude extract in a way that protein
    of interest always goes into the pellet or the
    supernatant.
  • Follow progress with functional assay

8
Lactate Dehydrogenase
  • NADH H Pyruvate ??NAD Lactate
  • Enzyme clears lactic acid from working muscles
  • The obvious source of enzyme is muscle tissue
    (heart skeletal muscle, HM, isomers)
  • We will assay for the enzymes ability to convert
    Pyruvate to Lactate

9
Begin with intact tissue
  • Disrupt (step45)
  • Blender, homoginizer
  • Remove debris (step7)
  • Centrifugation
  • Precipitate/concentrate (step 14-16)
  • Ammonium sulfate
  • Remove salt (step 22)
  • dialysis
  • Purify (next Lab)
  • Chromatography
  • Analyze (Part B and week 3 4)
  • Activity, molecular weight

10
Ammonium Sulfate pptpage 118
  • Has a wide range of application
  • Relies on fact that proteins loose solubility as
    concentration of salt is increased
  • Is characteristic of particular protein
  • Results in a partial purification of all proteins
    with similar solubility characteristics
  • Must determine amm sulf to precipitate your
    protein empirically.
  • Produces salt cuts

11
Salting in / Salting out
  • Salting IN
  • At low concentrations, added salt usually
    increases the solubility of charged
    macromolecules because the salt screens out
    charge-charge interactions.
  • So low salt prevents aggregation and therefore
    precipitation or crashing.
  • Salting OUT
  • At high concentrations added salt lowers the
    solubility of macromolecules because it competes
    for the solvent (H2O) needed to solvate the
    macromolecules.
  • So high salt removes the solvation sphere from
    the protein molecules and they come out of
    solution.

12
Kosmotrope vs. Chaotrope
  • Ammonium Sulfate
  • Increasing conc causes proteins to precipitate
    stably.
  • Kosmotropic ion stabilizing ion.
  • Urea
  • Increasing conc denatures proteins when they
    finally do precipitate, it is random and
    aggregated.
  • Chaotropic ion denaturing ion.

13
Dialysis
  • Passage of solutes through a semi-permeable
    membrane.
  • Pores in the dialysis membrane are of a certain
    size.
  • Protein stays in water, salts, protein
    fragments, and other molecules smaller than the
    pore size pass through.

14
Column Chromatography2nd Day Page 125
15
Gel Filtration
16
Principles of gel filtration (molecular sieving)
5. Estimate approximate molecular weight of
unknown proteins and/or protein
complexes using calibration curve with
pre-run standard proteins of known M.Wt. and
the following formula
1. Apply a mixture of proteins on a gel
filtration column (Sepharose,
Sephacryl, etc)
2. Collect fractions, typically 120 from
a 1.5x100 cm column. Do not change
buffer composition
3. High molecular weight macromolecules
(higher Stokes radius) elute first
Ve -Vo Vt - Vo
Ve elution volumeVo void volumeVt total
volume
Kav
106 Da 3x105 Da 105 Da 104 Da
4. Determine proteins in eluate using
suitable assay
Kav
Log M.Wt.
17
Ion Exchange
18
Affinity Chromatography
We will use bound Adenosine -5-monophosphate.
This is part Of NAD. LDH will Bind. Release
LDH by adding NADH
19
NAD
AMP
20
Affinity chromatography
  • Remember NADH is a co-substrate for lactate
    dehydrogenase.
  • We use AMP-Sepharose AMP is covalently bound to
    the affinity gel, which will not pass through the
    filter.
  • LDH binds to the AMP b/c it looks like half an
    NADH.
  • Thus LDH remains immobilized in the column until
    we ad NADH which binds tighter to the LDH.

21
Protein Purificationpage 130
Activity
A280
NADH
22
Protein Concentration
  • Lowry ( most cited reference in biology)
  • Color assay
  • A280
  • Intrinsic absorbance
  • Relies on aromatic amino acids
  • BCA page 133 137
  • Modification of Lowry increased sensitivity and
    consistency
  • Bradford
  • Shifts Amax of dye from 465nm to 595nm

23
A280 Page 114 131
  • Uses intrinsic absorbance
  • Detects aromatic residues
  • Resonating bonds
  • Depends on protein structure, native state and
    AA composition
  • Retains protein function

24
PAGE Apparatus (purity and MW)4th week Page 135,
142
25
Protein separation using SDS-PAGE(Laemmli system)
2. Run the electrophoresis until dye reaches
the end of the gel
1. Apply protein/dye samples into
polyacrylamide gel wells
Stackinggel
Resolvinggel
3. Remove the gel from the apparatus and
stain for proteins
26
SDS PAGE of Purification
  • Complete mix of proteins
  • High Salt
  • Ion exchange
  • Gel-filtratio
  • Affinity

10micrograms loaded in each lane
27
IMPORTANT
  • Do not throw away anything until you are certain
    you no longer need it
  • Biggest source of problem in this lab
  • Label everything clearly copy labels into lab
    book
  • Throwing out wrong fraction results in starting
    over
  • 3 days into experiment huge problem

28
Day 1 See Table C2-2 (page 117), Page 118-124.
138
29
Will follow Flow sheet Page 119
We will do only one NH4SO4 cut
Save 3 samples Will determine protein
concentration activity and purity
30
Will fill out this critical table as we proceed
page 138
Column C (Column A)(Column B) Column F
(Column A)(Column E) Column G Column C/Column
F Column B / Column E Column D Column
C/first value in Column C
31
Today. Page 118 (part of group)
  • Steps 1-5 Weigh muscle sample place in blender
    with 50ml ice cold buffer homogenize for 2
    minutes.
  • Steps 67 remove large debris by centrifugation
    Save Supernatant (remove 1ml (Microfuge tube) for
    later analysis).
  • Steps 9-13 Measure the volume of the supernatant
    determine amount of ammonium sulfate required for
    precipitation, weigh out 0.4 grams per/ml
    (NH4)2SO4

32
Today group 1 continued
  • Step14-16 Slowly add salt to gently stirred
    supernatant . Keep Cold!!See step 12
  • Step 17 Centrifuge precipitate to a pellet
  • Step 18-21 Save supernatant (1ml in microfuge
    tube). Suspend pellets in 5ml cold buffer
  • Step 22, 23 Add PMSF and place suspended pellet
    in dialysis tubing and give to TA

33
Today group 2
  • Set up standard assay as on page 122
  • Measure loss of absorbance as NADH is converted
    to NAD
  • Step 4 is similar to Kinetic curve you did for
    ADH (page 124) only reversed as measure loss of
    absorbance
  • Steps 8-12 You will determine the velocity of
    LDH catalyzed reaction by varying the
    concentration of LDH with constant substrate and
    cofactor. Be sure to adjust the amount of
    reaction buffer to give 3.2 ml final volume in
    each assay

34
Very Important Page 124
Blank without NADH
Blank with NADH
35
Today group 2 continued
  • You are establishing the assay conditions you
    will use next week to follow the purification of
    LDH. You must become proficient at this assay.

36
Flow chart 1B (page 122)
37
Spurious Vo MeasurementsSame as with ADH(this
is similar to your ADH exp)
38
Procedure (Page 122)
  • 1 Step 1-6. Will create a kinetic curve for LDH
    (adjust volume of buffer to make 3.2ml)
  • Similar to ADH
  • 2. Repeat kinetic curve with different
    concentrations of enzyme
  • This is protocol you will use as you purify LDH
  • Do this assay on the unknown samples from step
    one and 2a from group 1.

39
C2-3. Page 123
40
Next Week Column Chromatography
  • Due next time Prelab assignment for period 2 of
    LDH Purification
  • You really should write up or otherwise arrange
    what you did today as soon as possible. Do Not
    Trust Your Memory

41
Next lab
  • Need member of group to be here at 130 to begin
    washing column
  • Will need to measure absorbance at 280 to
    determine that contaminating protein is lost from
    column. Wash and measure until A280 is constant.

42
Strategy
  • For samples generated determine amount of
    protein (A280 ) and activity
  • Activity per microgram of protein s specific
    activity
  • You strive for maximal activity per unit of
    protein. (table C2-4 Column G, Page 138)

43
Will generate this elution profilePage 130
44
Will fill out this critical table as we proceed
page 138 (day 4)
Column C (Column A)(Column B) Column F
(Column A)(Column E) Column G Column C/Column
F Column B / Column E Column D Column
C/first value in Column C
45
This Lab
  • 4 lab periods
  • Prelab 12 points
  • Lab Report 50 points
  • First exam in period 4
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