ProteinDNARNA Concentration

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Protein/DNA/RNA Concentration

• Laurie Earls
• IGP methodology
• Fall 2002
• Laurie.Earls_at_Vanderbilt.edu
• 322-5268

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You are in Lab Late at Night and

• You have just purified a protein
• You have drawn serum from a goat
• You have just homogenized the livers of 35 adult
  male naked mole rats
• You have urine samples from patients that you
  suspect are hyperproteinuric

You ask yourself how much protein is in there?
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.Methods for Protein Determination

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Basic Principal of Protein Concentration
Determination
Make a standard curve using a protein of known
concentration, and use this curve to determine
the concentration of your protein
0.25mg/ml
0.50mg/ml
0.75mg/ml
1.00mg/ml
1.25mg/ml
1.50mg/ml
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Equipment for Protein Concentration Determination
Spectrophotometer
Less expensive
Requires more sample
Takes Longer
The math is up to you
Plate Reader
Expensive
Requires less sample
Fast
Some do the math for you
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The Data (regardless of method)
Sample
Concentration
Absorbance
Std 0
0 mg/mL
0.00
Std 0.25
0.25 mg/mL
0.142
Std 0.5
0.50 mg/mL
0.278
Std 0.75
0.75 mg/mL
0.385
Std 1.0
1.0 mg/mL
0.447
Std 1.25
1.25 mg/mL
0.576
Std 1.50
1.50 mg/mL
0.695
Std 1.75
1.75 mg/mL
0.821
unknown
0.535
unknown
0.572
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Creating and using a standard Curve
Y mX b
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Interpreting your standard Curve
r
0.2
0.97
r2 The fraction of the variation of Y that is
explained by the variation of X. The closer to 1
r2 is, the better predictor your line is.
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Using a plate reader
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Using a plate reader
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Absorbance for Determining Protein Concentration

• Aromatic amino acids absorb uv light at 280nm
• Peptide bonds absorb at 205nm
• The absorbance for a given amount of certain
  proteins is published
• There is a linear relationship between how much
  protein is present and how much light the
  solution will absorb

A280
Concentration(mg/mL)
a280 X pathlength
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Bradford Assay
?Probably most common
?Dye reacts with Arginine residues in acid pH and
produces a blue color which absorbs light at 595nm
?More color more protein
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Bradford Assay
Protein Concentration
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BCA Assay
1) Proteins reduce cupric (Cu II) ion to cuprous
(Cu III) ion
2) BCA interacts with the Cu I ion to produce a
purple color which absorbs light at 562nm
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BCA Assay
More protein more purple color
The Kit
Mix 50 parts reagent A with 1 part reagent B?
Turns green
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Tips to Painless Protein Assays

• Keep stocks of BSA standards in 20oC
• Use the same pipet and pipet carefully
• Keep some bradford reagent in light-proof
  container on your bench to avoid precipitation
• Avoid bubbles

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You are in Lab Late at Night and

• You want to send some DNA for sequencing
• You want to set up a ligation reaction for
  subcloning
• You want to transfect some DNA into HEK293 cells

You ask yourself how much DNA do I have?
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Methods for Determination of DNA concentration

• Ultraviolet absorbance at 260nm
• Fluorometry
• Agarose gel electrophoresis
• Parafilm spot method

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DNA Concentration by Abs260
? Dilute DNA in water or TE buffer
? Read Absorbance at 260 and 280
? Beers law A ebc
OD260 of 1.0 50mg/mL double stranded DNA
OD260 of 1.0 33mg/mL single stranded DNA
OD260 of 1.0 40mg/mL RNA
? So, if you use a 1cm pathlength cuvette, then
Conc. (mg/mL) Abs260 X 50 X dilution factor
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DNA concentration by agarose gel electrophoresis
Compare the intensity of your band with that of
bands on the ladder of known concentrations
Ex You load 2mL of DNA and see a band like this
You conclude that there are about 60ng of DNA
present divide by 2mL and you have 30ng/mL
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DNA Concentration by the Parafilm Spot Method
250ng/ml
500ng/ml
1mg/ml
100ng/ml
50ng/ml
unknown
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Mwahahahaaaa.Questions?