Protein Purification Lab C2Pages 101 to 142

Lab C.2

Four Periods

Protocol Page 118-142

Be sure to read theory starting page 104

Exam

• Exam March 14

• 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

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.

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

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

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

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

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, H&M, isomers)• We will assay for the enzymes ability to convert

Pyruvate to Lactate

Begin with intact tissue• Disrupt (step4&5)

– 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

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”

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.

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.

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.

Column Chromatography2nd Day Page 125

Gel Filtration

Principles of gel filtration (molecular sieving)

106 Da3x105 Da105 Da104 Da

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 Stoke’s radius) elute first

4. Determine proteins in eluate using suitable assay

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:

Kav = Ve -VoVt - Vo

Ve – elution volumeVo – void volumeVt – total volume

Kav

Log M.Wt.

Ion Exchange

Affinity Chromatography

We will use bound Adenosine-5’-monophosphate. This is part Of NAD+. LDH will Bind. Release LDH by adding NADH

NAD+

AMP

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.

Protein Purificationpage 130

A280

Activity

NADH

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

A280 Page 114 &131

• Uses intrinsic absorbance

• Detects aromatic residues – Resonating bonds

• Depends on protein structure, native state and AA composition

• Retains protein function

Protein separation using SDS-PAGE(Laemmli system)

Stackinggel

Resolvinggel

1. Apply protein/dye samples into polyacrylamide gel wells

2. Run the electrophoresis until dye reaches the end of the gel

3. Remove the gel from the apparatus and stain for proteins

SDS PAGE of Purification

1. Complete mix of proteins2. High Salt3. Ion exchange4. Gel-filtratio5. Affinity

10micrograms loaded in each lane

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

Will fill out this critical table as we proceed page 138

Table C.2-4. Enzyme Purification Table

Net volume(ml)

V0 units per

ml

V0 units

Total(an “amount”)

Protein content

(% of total)

Proteinconcentration

(mg/ml)

Net amount

of protein(mg)

Specific Activity(V0/mg

protein)

Step A B C D E F G

1. Cleared

2. (NH4)2SO4

Supernatant

3. diluted dialyzed sample/ solution placed on column

4. pooled peak tubes from column

Column C = (Column A)(Column B)Column F = (Column A)(Column E)Column G = Column C/Column F = Column B / Column EColumn D = Column C/first value in Column C

Very Important: Page 124

0

0.2

0.4

time (sec)60 120 1800

0

0.2

0.4

time (sec)60 120 1800

A B

observed

observed

extrapolated timecourse

Blank without NADH Blank with NADH

Spurious Vo MeasurementsSame as with ADH

(this is similar to your [ADH] exp)

0

0.2

0.4

0.6

0 15 30 45 60

more enzyme

time (sec)

A340

0

0.2

0.4

0.6

0 15 30 45 60

time (sec)

A340

A) Small [E] B) Increasing [E]

75

C2-3. Page 123Table C.2-3. Lactate Dehydrogenase Reaction Time Courses

Reading number

time(seconds)

A340 readings

50 ml sample

100 ml sample

200 ml sample

300 ml sample

400 ml sample

1 0

2 15

3 30

4 45

5 60

6 75

7 90

8 105

9 120

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

Next lab

• Need member of group to be here at 1:30 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.

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)

Will generate this elution profilePage 130

A280 V0

fraction (tube) number (approximate only)

NADH added

0

0 10 20 30 40 50 60 70 80

contaminant protein

LDH

Will fill out this critical table as we proceed page 138 (day 4)

Table C.2-4. Enzyme Purification Table

Net volume(ml)

V0 units per

ml

V0 units

Total(an “amount”)

Protein content

(% of total)

Proteinconcentration

(mg/ml)

Net amount

of protein(mg)

Specific Activity(V0/mg

protein)

Step A B C D E F G

1. Cleared

2. (NH4)2SO4

Supernatant

3. diluted dialyzed sample/ solution placed on column

4. pooled peak tubes from column

Column C = (Column A)(Column B)Column F = (Column A)(Column E)Column G = Column C/Column F = Column B / Column EColumn D = Column C/first value in Column C

This Lab

• 4 lab periods

• Prelab= 12 points

• Lab Report= 50 points

• First exam in period 4