5.2,Expert Experimentalist Rating:,A Heart as Strong as Iron”
Techniques Checklist,
Use of a centrifuge?
Equipment,
Disposable UV-Vis cuvettes ( 1-mL capacity)
Pipetmen,20 P,100 P,1000 P
Pipet tips
Eppendorf tubes (safe-lock)
Hot plate
Centrifuge
Boiling plate or rack to hold Eppendorf tubes
Large crystallizing dish
Goals,
From the CC-level experiment,you know the concentration of protein in your
sample,Now you will determine the concentration of iron in bovine heart cytochrome c,
Experiment Outline,
The Ferrozine Assay
Ferrozine is an iron-chelating agent,When it forms a complex with ferrous iron
(Fe
II
),it shows a characteristic UV-Vis absorption at 562 nm,By comparing the A
562
of
your sample to a calibration curve of iron standards,you will determine the concentration of
iron in your protein sample,
Solutions provided by your TA,
-Fe AA standard (AA = atomic absorption)
-Buffer - 25 mM MOPS,pH 7
-Ultrapure HNO
3
(5 M)
-75 mM Ascorbic acid solution
-10 mM Ferrozine solution
-Saturated ammonium acetate solution
35
WARNING NOTICE,The experiments described in these materials are potentially hazardous and require a high level
ofsafety training,special facilities and equipment,and supervision by appropriate individuals,You bear the sole
responsibility,liability,and risk for the implementation of such safety procedures and measures,MIT shall have no
responsibility,liability,or risk for the content or implementation of any of the material presented,Legal Notices
1,Preparation of Standards:
Prepare a fresh set of iron standards in 2 mL Eppendorf tubes,as illustrated
below,Carefully label each tube,Also fill 2 tubes with 300 μL of your
protein sample,
μL of Fe AA standard (99 μg/mL) μL of Buffer to add
0 300
6 294
12 288
18 282
24 276
Add 30 μL of ultrapure HNO
3
(5 M) to each standard and sample tube,
Place the closed Eppendorf tubes in a rack,and boil them for 30 minutes in a hot
water bath (a large Pyrex dish over a heating plate),
Centrifuge for 1-2 minutes,making sure the centrifuge is properly balanced.
Remove 300 μL of the supernatant liquid from each tube,and transfer to fresh
tubes (labeled!),
Add 1020 μL of distilled water,
Add 60 μL of 75 mM ascorbic acid,
Add 60 μL of 10 mM ferrozine,
Add 60 μL of saturated ammonium acetate,
Shake each tube and wait 10-15 minutes,(the solutions should become purplish in
color),
Transfer to a 1.5 mL cuvette,and determine the A
562
for each standard and your
two samples,
Generate a calibration curve of A
562
vs,[Fe] from your standards,
Determine the [Fe] in your unknown.
Results
To obtain your "EE Rating" in Protein Assays and Error Analysis,the line fit for
your standard curve must have a 0.995 correlation coefficient or higher,Additionally,the
absorbance values for your unknown samples must have a standard deviation of 0.035 or
less,Finally,you must determine the number of molecules of iron per molecule of protein,
36
Techniques Checklist,
Use of a centrifuge?
Equipment,
Disposable UV-Vis cuvettes ( 1-mL capacity)
Pipetmen,20 P,100 P,1000 P
Pipet tips
Eppendorf tubes (safe-lock)
Hot plate
Centrifuge
Boiling plate or rack to hold Eppendorf tubes
Large crystallizing dish
Goals,
From the CC-level experiment,you know the concentration of protein in your
sample,Now you will determine the concentration of iron in bovine heart cytochrome c,
Experiment Outline,
The Ferrozine Assay
Ferrozine is an iron-chelating agent,When it forms a complex with ferrous iron
(Fe
II
),it shows a characteristic UV-Vis absorption at 562 nm,By comparing the A
562
of
your sample to a calibration curve of iron standards,you will determine the concentration of
iron in your protein sample,
Solutions provided by your TA,
-Fe AA standard (AA = atomic absorption)
-Buffer - 25 mM MOPS,pH 7
-Ultrapure HNO
3
(5 M)
-75 mM Ascorbic acid solution
-10 mM Ferrozine solution
-Saturated ammonium acetate solution
35
WARNING NOTICE,The experiments described in these materials are potentially hazardous and require a high level
ofsafety training,special facilities and equipment,and supervision by appropriate individuals,You bear the sole
responsibility,liability,and risk for the implementation of such safety procedures and measures,MIT shall have no
responsibility,liability,or risk for the content or implementation of any of the material presented,Legal Notices
1,Preparation of Standards:
Prepare a fresh set of iron standards in 2 mL Eppendorf tubes,as illustrated
below,Carefully label each tube,Also fill 2 tubes with 300 μL of your
protein sample,
μL of Fe AA standard (99 μg/mL) μL of Buffer to add
0 300
6 294
12 288
18 282
24 276
Add 30 μL of ultrapure HNO
3
(5 M) to each standard and sample tube,
Place the closed Eppendorf tubes in a rack,and boil them for 30 minutes in a hot
water bath (a large Pyrex dish over a heating plate),
Centrifuge for 1-2 minutes,making sure the centrifuge is properly balanced.
Remove 300 μL of the supernatant liquid from each tube,and transfer to fresh
tubes (labeled!),
Add 1020 μL of distilled water,
Add 60 μL of 75 mM ascorbic acid,
Add 60 μL of 10 mM ferrozine,
Add 60 μL of saturated ammonium acetate,
Shake each tube and wait 10-15 minutes,(the solutions should become purplish in
color),
Transfer to a 1.5 mL cuvette,and determine the A
562
for each standard and your
two samples,
Generate a calibration curve of A
562
vs,[Fe] from your standards,
Determine the [Fe] in your unknown.
Results
To obtain your "EE Rating" in Protein Assays and Error Analysis,the line fit for
your standard curve must have a 0.995 correlation coefficient or higher,Additionally,the
absorbance values for your unknown samples must have a standard deviation of 0.035 or
less,Finally,you must determine the number of molecules of iron per molecule of protein,
36
